Editorial

No abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78238/1/21554_ftp.pd

Electron‐deficient p‐benzoyl‐l‐phenylalanine derivatives increase covalent chemical capture yields for protein–protein interactions

The photoactivatable amino acid p‐benzoyl‐l‐phenylalanine (pBpa) has been used for the covalent capture of protein–protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein–protein interactions. The analogs can be incorporated into live yeast and upon irradiation capture endogenous PPIs. Halogenated pBpas will extend the scope of PPIs that can be captured and expand the toolbox for mapping PPIs in their native environment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149350/1/pro3621.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149350/2/pro3621_am.pd

Transcriptional tools: Small molecules for modulating CBP KIX-dependent transcriptional activators

Previously it was demonstrated that amphipathic isoxazolidines are able to functionally replace the transcriptional activation domains of endogenous transcriptional activators. In addition, in vitro binding studies suggested that a key binding partner of these molecules is the CREB Binding Protein (CBP), more specifically the KIX domain within this protein. Here we show that CBP plays an essential role in the ability of isoxazolidine transcriptional activation domains to activate transcription in cells. Consistent with this model, isoxazolidines are able to function as competitive inhibitors of the activators MLL and Jun, both of which utilize a binding interaction with KIX to up-regulate transcription. Further, modification of the N2 side chain produced three analogs with enhanced potency against Jun-mediated transcription, although increased cytotoxicity was also observed. Collectively these small KIX-binding molecules will be useful tools for dissecting the role of the KIX domain in a variety of pathological processes. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 17–23, 2011.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78234/1/21548_ftp.pd

A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein–Protein Interactions

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein–protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post‐crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4–Gal80 transcriptional complex. Post‐crosslinking, the Gal4–Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin‐azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole‐cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.Using the bifunctional unnatural amino acid, BPKyne, we have developed a strategy to capture and directly label transient protein–protein interactions (PPIs) in their native environment. Click chemical functionalization post‐crosslinking with a biotin–azide probe enabled the isolation of transcriptional protein complexes from yeast cells. This amino acid will expand the toolbox for the discovery of new PPIs in live cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/1/cbic201600578.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/2/cbic201600578_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135955/3/cbic201600578-sup-0001-misc_information.pd

Covalent Chemical Cochaperones of the p300/CBP GACKIX Domain

The GACKIX activator binding domain has been a compelling target for small‐molecule probe discovery because of the central role of activator–GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context‐dependent function of activator–coactivator complexes. However, the dynamic and transient protein–protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules. An additional complication is that activator‐binding motifs, such as GACKIX, are found in multiple coactivators, making specificity difficult to attain. In this study, we demonstrate that the strategy of tethering can be used to rapidly discover highly specific covalent modulators of the dynamic PPIs between activators and coactivators. These serve as both ortho‐ and allosteric modulators, enabling the tunable assembly or disassembly of the activator–coactivator complexes formed between the KIX domain and its cognate activator binding partners MLL and CREB. The molecules maintain their function and selectivity, even in human cell lysates and in bacterial cells, and thus, will ultimately be highly useful probes for cellular studies.Joining forces: Reversible covalent modulators of the conformationally dynamic KIX coactivator are readily converted into irreversible inhibitors by replacement of the disulfide moiety. The irreversible inhibitors are effective and selective, even in human cell lysate.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146336/1/cbic201800173-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146336/2/cbic201800173.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146336/3/cbic201800173_am.pd

Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein–protein interaction

Protein–protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, ( p‐ benzoylphenylalanine (pBpa) and p ‐azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo‐crosslinking to capture novel PPIs and to characterize the interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 391–397, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102672/1/bip22395.pd

A Synthetic Loop Replacement Peptide That Blocks Canonical NFâ κB Signaling

Aberrant canonical NFâ κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many nonâ canonical NFâ κB functions. Here we show that a selective peptideâ based inhibitor of canonical NFâ κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a nonâ labile bond, shows an about 10â fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NFâ κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many proteinâ protein interactions mediated by such structures.A peptideâ based inhibitor of canonical NFâ κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a nonâ labile bond, shows an about 10â fold increased potency relative to the original inhibitor. The success of the synthetic replacement of a peptide loop suggests that the general strategy could be broadly useful for discovering modulators of many proteinâ protein interactions mediated by such structures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135096/1/anie201607990.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135096/2/anie201607990-sup-0001-misc_information.pd

Bifunctional Ligands Allow Deliberate Extrinsic Reprogramming of the Glucocorticoid Receptor

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor\u27s biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics

Garcinolic Acid Distinguishes Between GACKIX Domains and Modulates Interaction Networks

Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (KD 1 μM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX. Garcinolic acid engages full-length CBP in the context of the proteome and in doing so effectively inhibits KIX-dependent transcription in a leukemia model. As the most potent small-molecule KIX inhibitor yet reported, garcinolic acid represents an important step forward in the therapeutic targeting of CBP/p300

Influence of genetic factors on toluene diisocyanate-related symptoms: evidence from a cross-sectional study

Background: Toluene diisocyanate (TDI) is a highly reactive compound used in the production of, e. g., polyurethane foams and paints. TDI is known to cause respiratory symptoms and diseases. Because TDI causes symptoms in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility. Methods: Workers (N = 132) exposed to TDI and a non-exposed group ( N = 114) were analyzed for genotype (metabolising genes: CYP1A1*2A, CYP1A1*2B, GSTM1*O, GSTM3*B, GSTP1 1105V, GSTP1 A114V, GSTT1*O, MPO -463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, SULT1A1 R213H; immune-related genes: CCL5 -403, HLA-DQB1* 05, TNF-308, TNF-863) and symptoms of the eyes, upper and lower airways ( based on structured interviews). Results: For three polymorphisms: CYP1A1*2A, CYP1A1*2B, and TNF -308 there was a pattern consistent with interaction between genotype and TDI exposure status for the majority of symptoms investigated, although it did reach statistical significance only for some symptoms: among TDI-exposed workers, the CYP1A1 variant carriers had increased risk (CYP1A1*2A and eye symptoms: variant carriers OR 2.0 95% CI 0.68-6.1, p-value for interaction 0.048; CYP1A1*2B and wheeze: IV carriers OR = 12, 1.4-110, p-value for interaction 0.057). TDI-exposed individuals with TNF-308 A were protected against the majority of symptoms, but it did not reach statistical significance. In the non-exposed group, however, TNF -308 A carriers showed higher risk of the majority of symptoms ( eye symptoms: variant carriers OR = 2.8, 1.1-7.1, p-value for interaction 0.12; dry cough OR = 2.2, 0.69-7.2, p-value for interaction 0.036). Individuals with SULT1A1 213H had reduced risk both in the exposed and non-exposed groups. Other polymorphisms, showed associations to certain symptoms: among TDI-exposed, NAT1*10 carriers had a higher risk of eye symptoms and CCL5 -403 AG+AA as well as HLA-DQB1 *05 carriers displayed increased risk of symptoms of the lower airways. GSTM1, GSTM3 and GSTP1 only displayed effects on symptoms of the lower airways in the non-exposed group. Conclusion: Specific gene-TDI interactions for symptoms of the eyes and lower airways appear to exist. The results suggest different mechanisms for TDI- and non- TDI-related symptoms of the eyes and lower airways