Characterization of a cytokinin-binding protein locus in Mycobacterium tuberculosis.

Cytokinins are adenine-based hormones that have been well-characterized in plants but are also made by bacteria, including the human-exclusive pathogen Mycobacterium tuberculosis . In M. tuberculosis , cytokinins activate transcription of an operon that affects the bacterial cell envelope. In plants, cytokinins are broken down by dedicated enzymes called cytokinin oxidases into adenine and various aldehydes. In proteasome degradation-deficient M. tuberculosis , the cytokinin-producing enzyme Log accumulates, resulting in the buildup of at least one cytokinin-associated aldehyde. We therefore hypothesized that M. tuberculosis encodes one or more cytokinin oxidases. Using a homology-based search for homologs of a plant cytokinin oxidase, we identified Rv3719 and a putative cytokinin-specific binding protein, Rv3718c. Deletion of the locus encoding these proteins did not have a measurable effect on in vitro growth. Nonetheless, Rv3718c bound a cytokinin with high specificity. Our data thus support a model whereby cytokinins play one or more roles in mycobacterial physiology. IMPORTANCE: Numerous bacterial species encode cytokinin-producing enzymes, the functions of which are almost completely unknown. This work contributes new knowledge to the cytokinin field for bacteria, and also revealed further conservation of cytokinin-associated proteins between plants and prokaryotes.

Targeting allostery in the Dynein motor domain with small molecule inhibitors.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Affinity purification probes of potential use to investigate the endogenous Hsp70 interactome in cancer.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.