Chemical compounds that interfere with microtubules such as the vinca alkaloids and taxanes are important chemotherapeutic agents for the treatment of cancer. As our knowledge of microtubule-targeting drugs increases, we realize that the mechanism underlying the anti-cancer activity of these agents may mainly lie in their inhibitory effects on spindle microtubule dynamics, rather than in their effects on microtubule polymer mass. There is increasing evidence showing that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting cell cycle progression at mitosis and eventually leading to apoptotic cell death. The effectiveness of microtubule-targeting drugs for cancer therapy has been impaired by various side effects, notably neurological and hematological toxicities. Drug resistance is another notorious factor that thwarts the effectiveness of these agents, as with many other cancer chemotherapeutics. Several new microtubule-targeting agents have shown potent activity against the proliferation of various cancer cells, including cells that display resistance to the existing microtubule-targeting drugs. Continued investigation of the mechanisms of action of microtubule-targeting drugs, development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance may provide more effective therapeutic options for cancer patients.

Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Noscapine, a microtubule-interfering agent, has been shown to arrest mitosis, to induce apoptosis, and to have potent antitumor activity. We report herein that two brominated derivatives of noscapine, 5-bromonoscapine (5-Br-nosc) and reduced 5-bromonoscapine (Rd 5-Br-nosc), have higher tubulin binding activity than noscapine and affect tubulin polymerization differently from noscapine. In addition, they are able to arrest cell cycle progression at mitosis at concentrations much lower than noscapine. Interestingly, whereas noscapine-arrested cells have nearly normal bipolar spindles, cells arrested by 5-Br-nosc and Rd 5-Br-nosc form multipolar spindles. Nevertheless, noscapine and the two derivatives all affect the attachment of chromosomes to spindle microtubules and they impair the tension across paired kinetochores to similar degrees. 5-Br-nosc and Rd 5-Br-nosc are also more active than noscapine in inhibiting the proliferation of various human cancer cells, including those that are resistant to paclitaxel and epothilone. Our results thus indicate a great potential for the use of 5-Br-nosc and Rd 5-Br-nosc both as biological tools for studying microtubule-mediated processes and as chemotherapeutic agents for the treatment of human cancers.

We have previously identified the opium alkaloid noscapine as a microtubule interacting agent that binds stoichiometrically to tubulin and alters its conformation. Here we show that, unlike many other microtubule inhibitors, noscapine does not significantly promote or inhibit microtubule polymerization. Instead, it alters the steady-state dynamics of microtubule assembly, primarily by increasing the amount of time that the microtubules spend in an attenuated (pause) state. Further studies reveal that even at high concentrations, noscapine does not alter the tubulin polymer/monomer ratio in HeLa cells. Cells treated with noscapine arrest at mitosis with nearly normal bipolar spindles. Strikingly, although most of the chromosomes in these cells are aligned at the metaphase plate, the rest remain near the spindle poles, both of which exhibit loss of tension across kinetochore pairs. Furthermore, levels of the spindle checkpoint proteins Mad2, Bub1, and BubR1 decrease by 138-, 3.7-, and 3.9-fold, respectively, at the kinetochore region upon chromosome alignment. Our results thus suggest that an exquisite control of microtubule dynamics is required for kinetochore tension generation and chromosome alignment during mitosis. Our data also support the idea that Mad2 and Bub1/BubR1 respond to kinetochore-microtubule attachment and/or tension to different degrees.

The transcription factor KLF5 plays an important role in human carcinogenesis. In epithelial cells, the KLF5 protein is tightly regulated by the ubiquitin-proteasome pathway. To better understand the mechanisms for the regulation of KLF5 protein, we identified and characterized an E3 ubiquitin ligase for KLF5, i.e. WWP1. We found that WWP1 formed a protein complex with KLF5 in vivo and in vitro. Furthermore, WWP1 mediated the ubiquitination and degradation of KLF5, and the catalytic cysteine residue of WWP1 is essential for its function. A PY motif in a transactivation domain of KLF5 is necessary for its interaction with WWP1. Finally, WWP1 was amplified and overexpressed in some cancer cell lines from the prostate and breast, which negatively regulated the function of KLF5 in gene regulation. These findings not only established WWP1 as an E3 ubiquitin ligase for KLF5, they also further implicated the KLF5 pathway in human carcinogenesis.

Mutations in Parkin, an E3 ubiquitin ligase that regulates protein turnover, represent one of the major causes of familial Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons and impaired mitochondrial functions. The underlying mechanism by which pathogenic Parkin mutations induce mitochondrial abnormality is not fully understood. Here, we demonstrate that Parkin interacts with and subsequently ubiquitinates dynamin-related protein 1 (Drp1), for promoting its proteasome-dependent degradation. Pathogenic mutation or knockdown of Parkin inhibits the ubiquitination and degradation of Drp1, leading to an increased level of Drp1 for mitochondrial fragmentation. These results identify Drp1 as a novel substrate of Parkin and suggest a potential mechanism linking abnormal Parkin expression to mitochondrial dysfunction in the pathogenesis of Parkinson disease. Parkinson disease (PD)4 is one of the most common neurodegenerative diseases affecting over 2% populations over 65 years of age. It is classically characterized by the loss of dopaminergic neurons that project from the midbrain substantia nigra to the striatum (1, 2). Although the loss of dopaminergic neurons is responsible for the symptom of movement disorder in PD, it is now clear that other types of neurons throughout the brain are also affected in the disease (3, 4). The identification of genes linking to PD has greatly advanced our understanding of the molecular pathogenesis of the disease (5-8). Mutations in Parkin represent one of major causes for early onset of familial PD (9 -11). Parkin is an E3 ubiquitin ligase that contains two ring finger domains (12-15). A handful of substrates have been identified, including Parkin itself and CDCrel-1, synphilin-1, Pael-R, glycosylated ␣-synuclein, FBP1 (far upstream elementbinding protein 1), and the RNA-processing protein subunit p38/AIMP2 (16 -19). A putative mechanism by which mutations of Parkin cause PD would be abnormal accumulation and aggregation of the above substrates due to insufficient E3 ligase activity for ubiquitin-proteasome-dependent protein turnover (18,20,21). Surprisingly, only p38/AIMP2 and FBP1 were found to be accumulated in the brain samples of PD patients or in Parkin knock-out mice (16,17,19). Even though a number of the putative substrates have been identified, the causative link between these substrates and the PD pathogenesis remains not fully understood.Over the past few decades, accumulating evidence has suggested that mitochondrial dysfunction and the resulting oxidative damage are associated with PD. This is supported by a large number of reports demonstrating impaired mitochondrial functions in PD patients (22)(23)(24)(25)(26). Mitochondria undergo frequent fission, fusion, and redistribution throughout the cytoplasm in response to the energy needs (27,28). Either disruption of the fusion process or enhancement of the fission process renders the normal, tubular network of mitochondria to fragment into short rods or spheres (29). Abnormal...

The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1–8. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10–16. Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20. However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions. Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation. Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.