An engineered cell line with a hRpn1-attached handle to isolate proteasomes.

Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.

Prolonged mitosis results in structurally aberrant and over-elongated centrioles.

Centrioles are precisely built microtubule-based structures that assemble centrosomes and cilia. Aberrations in centriole structure are common in tumors, yet how these aberrations arise is unknown. Analysis of centriole structure is difficult because it requires demanding electron microscopy. Here we employ expansion microscopy to study the origins of centriole structural aberrations in large populations of human cells. We discover that centrioles do not have an elongation monitoring mechanism, which renders them prone to over-elongation, especially during prolonged mitosis induced by various factors, importantly including supernumerary centrioles. We identify that mitotic centriole over-elongation is dependent on mitotic Polo-like kinase 1, which we uncover as a novel regulator of centriole elongation in human cycling cells. While insufficient Plk1 levels lead to the formation of shorter centrioles lacking a full set of microtubule triplets, its overactivity results in over-elongated and structurally aberrant centrioles. Our data help explain the origin of structurally aberrant centrioles and why centriole numerical and structural defects coexist in tumors.

AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on T. brucei using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.

Kinase scaffold repurposing for neglected disease drug discovery: discovery of an efficacious, lapatinib-derived lead compound for trypanosomiasis.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei . Because drugs in use against HAT are toxic and require intravenous dosing, new drugs are needed. Initiating lead discovery campaigns by using chemical scaffolds from drugs approved for other indications can speed up drug discovery for neglected diseases. We demonstrated recently that the 4-anilinoquinazolines lapatinib (GW572016, 1) and canertinib (CI-1033) kill T. brucei with low micromolar EC50 values. We now report promising activity of analogues of 1, which provided an excellent starting point for optimization of the chemotype. Our compound optimization that has led to synthesis of several potent 4-anilinoquinazolines, including NEU617, 23a, a highly potent, orally bioavailable inhibitor of trypanosome replication. At the cellular level, 23a blocks duplication of the kinetoplast and arrests cytokinesis, making it a new chemical tool for studying regulation of the trypanosome cell cycle.