During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase.

Summary Tumor necrosis factor α (TNFα) has both positive and negative roles in human disease. In certain cancers, TNFα is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNFα antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNFα-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNFα stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated TAK1 that binds within the ATP binding pocket, yet is non-competitive, and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNFα-induced cell death, with general implications for cancer and autoimmune disease treatment.

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled–coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled–coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30 -40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.

Background: Pregnancy promotes physiological adaptations throughout the body mediated by the female sex hormones. Results: Pregnancy promotes switching of skeletal muscle to a glycolytic phenotype through the smoothelin-like protein 1 transcriptional cofactor. Conclusion: Deletion of SMTNL1 is able to mimic the effect of pregnancy in mice. Significance: Novel mechanism to explain insulin resistance during pregnancy.

Summary Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

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Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.