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The Bcl-2-associated death promoter BAD is a prognostic indicator for good clinical outcome of breast cancer patients; however, whether BAD affects breast cancer biology is unknown. Here we showed that BAD increased cell growth in breast cancer cells through two distinct mechanisms. Phosphorylation of BAD at S118 increased S99 phosphorylation, 14-3-3 binding and AKT activation to promote growth and survival. Through a second, more prominent pathway, BAD stimulated mitochondrial oxygen consumption in a novel manner that was downstream of substrate entry into the mitochondria. BAD stimulated complex I activity that facilitated enhanced cell growth and sensitized cells to apoptosis in response to complex I blockade. We propose that this dependence on oxidative metabolism generated large but nonaggressive cancers. This model identifies a non-canonical role for BAD and reconciles BAD-mediated tumor growth with favorable outcomes in BAD-high breast cancer patients.

Breast cancer patients are commonly treated with taxane (e.g. docetaxel) chemotherapy, despite poor outcomes and eventual disease relapse. We previously identified the Bcl-2-associated death promoter (BAD) as a prognostic indicator of good outcome in taxane-treated breast cancer patients. We also demonstrated that BAD expression in human breast carcinoma cells generated larger tumors in mouse xenograft models. These paradoxical results suggest that BAD-expressing tumors are differentially sensitive to taxane treatment. We validated this here and show that docetaxel therapy preferentially reduced growth of BAD-expressing xenograft tumors. We next explored the cellular mechanism whereby BAD sensitizes cells to docetaxel. taxanes are microtubule inhibiting agents that cause cell cycle arrest in mitosis whereupon the cells either die in mitosis or aberrantly exit (mitotic slippage) and survive as polyploid cells. In response to docetaxel, BAD-expressing cells had lengthened mitotic arrest with a higher proportion of cells undergoing death in mitosis with decreased mitotic slippage. Death in mitosis was non-apoptotic and not dependent on Bcl-XL interaction or caspase activation. Instead, cell death was necroptotic, and dependent on RoS. these results suggest that BAD is prognostic for favourable outcome in response to taxane chemotherapy by enhancing necroptotic cell death and inhibiting the production of potentially chemoresistant polyploid cells. Triple-negative breast cancer patients receive taxane chemotherapy, such as docetaxel (Taxotere ®), as standard first-line treatment despite an overall poor prognosis, high rate of relapse, and adverse effects 1. While multiple causes of cellular taxane resistance are known, these have not yet provided clinical markers to guide taxane therapy decisions 2-4. Understanding the molecular mechanisms that mediate outcome to taxane therapy may identify predictive biomarkers and novel therapeutic targets. The Bcl-2 family member BAD (Bcl-2-associated death promoter) is a prognostic indicator for good clinical outcome of taxane-treated breast cancer patients 5. BAD modulates breast cancer cell proliferation and tumor progression by regulating cell cycle progression 6,7. Thus, understanding how BAD better predicts patient outcome could aid in understanding docetaxel chemoresistance. Taxanes are anti-mitotic drugs that perturb microtubule dynamics, leading to chronic activation of the spindle assembly checkpoint and inhibition of the anaphase promoting complex that delays the degradation of cyclin B1 and inhibits mitotic exit 8. Ideally, this aberrant mitotic arrest initiates cell death in mitosis by facilitating the accumulation of a caspase-dependent death signal 9. Often, however, cells degrade sufficient cyclin B1 prior to full activation of apoptotic caspases, and cells slip out of mitosis in the absence of cytokinesis and enter G1 as polyploid cells. These polyploid cells have differential fates of G1 arrest, post-mitotic death, or continued cell cycle progression 10,11. The su...

Nucleotide-binding and oligomerization domain like receptors (NLR) are pattern recognition receptors used to provide rapid immune response by detecting intracellular pathogen-associated molecules. Loss of NLR activity is implicated in genetic disorders, disruption of adaptive immunity, and chronic inflammation. One NLR protein, NOD2, is frequently mutated in Crohn's disease (CD), which is an inflammatory disease of the gastrointestinal tract. Three commonly occurring CD-associated NOD2 mutations, R702W, G908R, and L1007fs, are clustered near the regulatory domain, leucine rich region (LRR), and lowers the activity of NOD2 in response to muramyl dipeptide (MDP). As LRR is also the ligand binding domain, this suggests that the mutations either affect the binding of MDP or how the molecule responds to ligand binding. To model the role of R702 in ligand-dependent activation of NOD2, we used homology modeling to map the residue R702 to the interface between the oligomerization domain and LRR. We show that a peptide derived from NOD2(697-718) binds LRR in vitro, and upon co-expressing or importing the peptide into HEK293 expressing NOD2, there is an increase in the MDP-dependent NOD2 activity. The study thus suggests that the R702W mutation interferes with the conformational changes needed for MDP binding and activation. J. Cell. Biochem. 118: 1227-1238, 2017. © 2016 Wiley Periodicals, Inc.

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. They are particularly useful to test hypotheses motivated by biochemical and structural studies. We used yeast display to engineer monobodies that bind a defined surface patch on the mitogen activated protein kinase (MAPK), Erk-2. The targeted area (“CD” domain) is known to control the specificity and catalytic efficiency of phosphorylation by the kinase by binding a linear peptide (“D” peptide) on substrates and regulators. An inhibitor of the interaction should thus be useful for regulating Erk-2 signaling in vivo. Although the CD domain constitutes only a small percentage of the surface area of the enzyme (~ 5%), sorting a yeast displayed monobody library with wild type (wt) Erk-2 and a rationally designed mutant led to isolation of high affinity clones with desired epitope specificity. The engineered binders inhibited the activity of Erk-2 in vitro and in mammalian cells. Furthermore, they specifically inhibited the activity of Erk-2 orthologs in yeast and suppressed a mutant phenotype in round worms caused by overactive MAPK signaling. The study therefore shows that positive and negative screening can be used to bias the evolution of epitope specificity and predictably design inhibitors of biologically relevant protein-protein interaction.

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein-protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfide to covalently crosslink the subunits of a complex, so that the disulfide-trapped complex can be displayed on the yeast surface for detection and analysis. Transient protein complexes are difficult to display on the yeast surface, since they may dissociate before they can be detected due to a long induction period in yeast. To this end, we show that three different quaternary structures with the subunit dissociation constant K(d) approximately 0.5-20 microM, the antibody variable domain (Fv), the IL-8 dimer, and the p53-MDM2 complex, cannot be displayed on the yeast surface as a noncovalent complex. However, when we introduce an interchain disulfide between the subunits, all three systems are efficiently displayed on the yeast surface, showing that disulfide trapping can help display protein complexes that cannot be displayed otherwise. We also demonstrate that a disulfide forms only between the subunits that interact specifically, the displayed complexes exhibit functional characteristics that are expected of wt proteins, the mutations that decrease the affinity of subunit interaction also reduce the display efficiency, and most of the disulfide stabilized complexes are formed within the secretory pathway during export to the surface. Disulfide crosslinking is therefore a convenient way to study weak protein association in the context of yeast display.

In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.The understanding of the regulation of secretion from exocrine organs, such as the pancreas and parotid gland, has been facilitated by the availability of biochemical assays for the secretory product of these glands (i.e., amylase). Using amylase assays, the influence of neurotransmitters, Ca2+ and cyclic nucleotides on secretory activity has been assessed (1-3). Other exocrine organs do not contain wellcharacterized secretory products and, in addition, may be cellularly heterogeneous. Studies of cellular secretory mechanisms in these organs have been handicapped by the lack of biochemical markers for individual cell types. Among such organs are the stomach, duodenum, and trachea.In the trachea (Fig. 1), goblet, gland, and ciliated epithelial cells generate a mixture of macromolecules, which constitutes '=5% by weight of the complex tracheal secretion known as "mucus." The composition of mucous glycoproteins has been partially described (4-9). Properties of the secretion including volume (10), sulfur content (11, 12), total protein (10), and molecular weight (13) can be altered by various forms of autonomic nervous system stimulation. A priori considerations suggest that these variations may arise by ,um, toluidine blue. (Bar = 10 ,m.) d...

Because streptavidin is a homotetramer, it can bind multiple biotinylated ligands and cause target aggregation. To allow biotin detection without clustering, we previously engineered monomeric streptavidin (mSA) that is structurally similar to a single streptavidin subunit. Introducing the S25H mutation near the binding site increases the biotin dissociation half-life t 1/2 to 83 minutes. The slowly dissociating mutant, mSA2, is useful in imaging studies because it allows stable labeling of biotinylated targets. We show that mSA2 conjugated with Alexa 488 binds biotinylated receptors on HEK293 with high specifi city, and bound mSA2-Alexa488 does not dissociate signifi cantly during an imaging study lasting 50 minutes. As a structural monomer, mSA2 can be fused to other proteins to create bifunctional molecules. We tested the use of mSA2 in proximity dependent biotinylation, in which mSA2 is fused to a peptide or a protein that binds a protein of interest (POI) and is used to recruit photoactivatable biotin (PA-biotin) to the target molecule. Once the resulting cluster of interacting proteins is subjected to UV-initiated distance-dependent biotinylation, subsequent affi nity purifi cation of biotinylated proteins on streptavidin beads can identify protein molecules that interact with POI. In addition to proteins that directly interact with the mSA2 fusion, mSA2 also induces biotinylation of other proteins that are associated through a series of noncovalent interactions. We show that mSA2 fused to an antibody recognition domain can be recruited to the kinase Erk-2 using a commercially available antibody and induce biotinylation of a known Erk-2 substrate, GST-Elk-1. Therefore, mSA2 can be used to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions. INNOVATIONStreptavidin is used broadly in biotechnology applications that require high-affi nity interaction with biotinylated ligands. Th e streptavidin-biotin system is versatile and easy to work with, which has contributed to its popular use. For example, biotin can be introduced using chemical and enzymatic reactions 1 , and biotinylated targets are readily detected using streptavidin. Because biotin is a small, chemically inert, hydrophilic molecule, biotinylation does not signifi cantly disrupt the native structure or stability of the target molecule or introduce a reactive group that results in non-specifi c interactions. Also, streptavidin has high thermal and aqueous stability, which are important to ensure the molecule retains function under many experimental conditions. Because streptavidin can be modifi ed with fl uorophores or enzymes without loss of function, it can be used for labeling biotinylated targets for detection and readout. Several excellent reviews on the applications of the streptavidin-biotin technology exist and may be referenced for additional information 2-5 .While achieving the highest sensitivity is essential for some applications, optimizing properties other than the affi nity of interaction may ...