UA62784 Is a Cytotoxic Inhibitor of Microtubules, not CENP-E

SummaryA recent screen for compounds that selectively targeted pancreatic cancer cells isolated UA62784. We found that UA62784 inhibits microtubule polymerization in vitro. UA62784 interacts with tubulin dimers ten times more potently than colchicine, vinblastine, or nocodazole. Competition experiments revealed that UA62784 interacts with tubulin at or near the colchicine-binding site. Nanomolar doses of UA62784 promote the accumulation of mammalian cells in mitosis, due to aberrant mitotic spindles, as shown by immunofluorescence and live cell imaging. Treatment of cancerous cell lines with UA62784 is lethal, following activation of apoptosis signaling. By monitoring mitotic spindle perturbations and apoptosis, we found that the effects of UA62784 and of some known microtubule-depolymerizing drugs are additive. Finally, high content screening of H2B-GFP HeLa cells revealed that low doses of UA62784 and vinblastine potentiate each other to inhibit proliferation

Toxin-Based Therapeutic Approaches

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin

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MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function

The structure of human neuronal Rab6B in the active and inactive form

The Rab small G-protein family plays important roles in eukaryotes as regulators of vesicle traffic. In Rab proteins, the hydrolysis of GTP to GDP is coupled with association with and dissociation from membranes. Conformational changes related to their different nucleotide states determine their effector specificity. The crystal structure of human neuronal Rab6B was solved in its 'inactive' (with bound MgGDP) and 'active' (MgGTP gamma S-bound) forms to 2.3 and 1.8 angstrom, respectively. Both crystallized in space group P2(1)2(1)2(1), with similar unit-cell parameters, allowing the comparison of both structures without packing artifacts. Conformational changes between the inactive GDP and active GTP-like state are observed mainly in the switch I and switch II regions, confirming their role as a molecular switch. Compared with other Rab proteins, additional changes are observed in the Rab6 subfamily-specific RabSF3 region that might contribute to the specificity of Rab6 for its different effector proteins

Certain protein transducing agents convert translocated proteins into cell killers.

The majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution

Otubains: a new family of cysteine proteases in the ubiquitin pathway

The modification of cellular proteins by ubiquitin (Ub) is an important event that underlies protein stability and function in eukaryotes. Protein ubiquitylation is a dynamic and reversible process; attached Ub can be removed by deubiquitylating enzymes (DUBs), a heterogeneous group of cysteine proteases that cleave proteins precisely at the Ub–protein bond. Two families of DUBs have been identified previously. Here, we describe new, highly specific Ub iso-peptidases, that have no sequence homology to known DUBs, but which belong to the OTU (ovarian tumour) superfamily of proteins. Two novel proteins were isolated from HeLa cells by affinity purification using the DUB-specific inhibitor, Ub aldehyde (Ubal). We have named these proteins otubain 1 and otubain 2, for OTU-domain Ubal-binding protein. Functional analysis of otubains shows that the OTU domain contains an active cysteine protease site

L'Alerte : journal indépendant : politique, littéraire et commercial

24 septembre 19061906/09/24 (A10)-1906/09/24