The tubulin database: Linking mutations, modifications, ligands and local interactions.

Microtubules are polymeric filaments, constructed of α-ß tubulin heterodimers that underlie critical subcellular structures in eukaryotic organisms. Four homologous proteins (γ-, δ-, ε- and ζ-tubulin) additionally contribute to specialized microtubule functions. Although there is an immense volume of publicly available data pertaining to tubulins, it is difficult to assimilate all potentially relevant information across diverse organisms, isotypes, and categories of data. We previously assembled an extensive web-based catalogue of published missense mutations to tubulins with >1,500 entries that each document a specific substitution to a discrete tubulin, the species where the mutation was described and the associated phenotype with hyperlinks to the amino acid sequence and citation(s) for research. This report describes a significant update and expansion of our online resource (TubulinDB.bio.uci.edu) to nearly 18,000 entries. It now encompasses a cross-referenced catalog of post-translational modifications (PTMs) to tubulin drawn from public datasets, primary literature, and predictive algorithms. In addition, tubulin protein structures were used to define local interactions with bound ligands (GTP, GDP and diverse microtubule-targeting agents) and amino acids at the intradimer interface, within the microtubule lattice and with associated proteins. To effectively cross-reference these datasets, we established a universal tubulin numbering system to map entries into a common framework that accommodates specific insertions and deletions to tubulins. Indexing and cross-referencing permitted us to discern previously unappreciated patterns. We describe previously unlinked observations of loss of PTM sites in the context of cancer cells and tubulinopathies. Similarly, we expanded the set of clinical substitutions that may compromise MAP or microtubule-motor interactions by collecting tubulin missense mutations that alter amino acids at the interface with dynein and doublecortin. By expanding the database as a curated resource, we hope to relate model organism data to clinical findings of pathogenic tubulin variants. Ultimately, we aim to aid researchers in hypothesis generation and design of studies to dissect tubulin function.

Diaryl and heteroaryl sulfides: synthesis via sulfenyl chlorides and evaluation as selective anti-breast-cancer agents.

A mild protocol for the synthesis of diaryl and heteroaryl sulfides is described. In a one-pot procedure, thiols are converted to sulfenyl chlorides and reacted with arylzinc reagents. This method tolerates functional groups including aryl fluorides and chlorides, ketones, as well as N-heterocycles including pyrimidines, imidazoles, tetrazoles, and oxadiazoles. Two compounds synthesized by this method exhibited selective activity against the MCF-7 breast cancer cell line in the micromolar range.

Stereospecific nickel-catalyzed cross-coupling reactions of alkyl Grignard reagents and identification of selective anti-breast-cancer agents.

Alkyl Grignard reagents that contain ß-hydrogen atoms were used in a stereospecific nickel-catalyzed cross-coupling reaction to form C(sp(3))-C(sp(3)) bonds. Aryl Grignard reagents were also utilized to synthesize 1,1-diarylalkanes. Several compounds synthesized by this method exhibited selective inhibition of proliferation of MCF-7 breast cancer cells.

The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis.

OBJECTIVES: In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection. METHODS: Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae prior to infection, or by adding Pep-1 to the medium at varying times after infection, and assaying for inhibition of inclusion formation. RESULTS: Pep-1 had a concentration-dependent effect on chlamydial growth with 100% inhibition of inclusion formation at 8 mg/L peptide. There was a window of susceptibility during the chlamydial developmental cycle with a maximal effect when treatment was begun within 12 h of infection. Pep-1 treatment caused a severe reduction in the production of infectious progeny even when started later, when the effect on inclusion formation was minimal. Furthermore, electron micrographs showed a paucity of progeny elementary bodies (EBs) in the inclusion. In contrast, pre-incubation of EBs with Pep-1 prior to infection did not affect inclusion formation. Taken together, these findings indicate that the antichlamydial effect was specific for the intracellular stage of chlamydial infection. By comparison, Pep-1 had no antimicrobial activity against Escherichia coli and Staphylococcus aureus or the obligate intracellular parasite, Toxoplasma gondii. CONCLUSIONS: Pep-1 has antichlamydial activity by preventing intracellular chlamydial growth and replication but has no effect on extracellular chlamydiae.

FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells.

Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activation. Here we show that T cells lacking FADD or casp8 activity are subject to hyperactive autophagic signaling and subvert a cellular survival mechanism into a potent death process. T cell autophagy, enhanced by mitogenic signaling, recruits casp8 through interaction with FADD:Atg5-Atg12 complexes. Inhibition of autophagic signaling with 3-methyladenine, dominant-negative Vps34, or Atg7 shRNA rescued T cells expressing a dominant-negative FADD protein. The necroptosis inhibitor Nec-1, which blocks receptor interacting protein kinase 1 (RIP kinase 1), also completely rescued T cells lacking FADD or casp8 activity. Thus, while autophagy is necessary for rapid T cell proliferation, our findings suggest that FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing RIPK1-dependent necroptotic cell death. Thus, linkage of FADD and casp8 to autophagic signaling intermediates is essential for rapid T cell clonal expansion and may normally serve to promote caspase-dependent apoptosis under hyperautophagic conditions, thereby averting necrosis and inflammation in vivo.

Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii.

The tachyzoite stage of the protozoan parasite Toxoplasma gondii has two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical polar ring for the subpellicular microtubules and spindle pole plaques/centrioles for the spindle microtubules. When replicating tachyzoites were treated with 0.5 microM oryzalin or 1.0 mM colchicine they retained the capacity to form a spindle and undergo nuclear division. Although these parasites could complete budding, they lost the bulk of their subpellicular microtubules and the ability to reinvade host cells. Both nascent spindle and subpellicular microtubules were disrupted in 2.5 microM oryzalin or 5.0 mM colchicine. Under these conditions, parasites grew in size and replicated their genome but were incapable of nuclear division. After removal from 0.5 microM oryzalin, Toxoplasma tachyzoites were able to restore normal subpellicular microtubules and a fully invasive phenotype. When oryzalin was removed from Toxoplasma tachyzoites treated with 2.5 microM drug, the parasites attempted to bud as crescent-shaped tachyzoites. Because the polyploid nuclear mass could not be correctly segregated, many daughter parasites lacked nuclei altogether although budding and scission from the maternal mass was able to be completed. Multiple MTOCs permit Toxoplasma tachyzoites to control nuclear division independently from cell polarity and cytokinesis. This unusual situation grants greater cell cycle flexibility to these parasites but abolishes the checks for coregulation of nuclear division and cytokinesis found in other eukaryotes.