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

Information for handover management in heterogeneous networks: data representation,languages and integrated platforms

Due to the convergence of radio, television, telephony and Internet areas, the mobility of users, the ubiquity of services, and the development of new technologies to unify access provision, the interaction between providers and users will be required for access on demand in heterogeneous environments. This interaction should allow, in addition to seamless handovers, the negotiation based on technical requirements and user's desires during handover decision processes. The central part of the information being exchanged between the access provider's attachment points and user's devices should be a uniform and common structure that models the handover management information, in terms of what the information represents their semantic meanings and relationships. This work presents a set of ontologies, for this purpose, employed during handover decision processes, in integrated networking platforms for access on demand. A case study is presented, which demonstrates how a service could be integrated in two different platforms for such environment

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

Help-seeking for genitourinary symptoms: a mixed methods study from Britain's Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3)

Objectives Quantify non-attendance at sexual health clinics and explore help-seeking strategies for genitourinary symptoms. Design Sequential mixed methods using survey data and semistructured interviews. Setting General population in Britain. Participants 1403 participants (1182 women) from Britain’s Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3; undertaken 2010–2012), aged 16–44 years who experienced specific genitourinary symptoms (past 4 weeks), of whom 27 (16 women) who reported they had never attended a sexual health clinic also participated in semistructured interviews, conducted May 2014–March 2015. Primary and secondary outcome measures From survey data, non-attendance at sexual health clinic (past year) and preferred service for STI care; semistructured interview domains were STI social representations, symptom experiences, help-seeking responses and STI stigma. Results Most women (85.9% (95% CI 83.7 to 87.9)) and men (87.6% (95% CI 82.3 to 91.5)) who reported genitourinary symptoms in Natsal-3 had not attended a sexual health clinic in the past year. Around half of these participants cited general practice (GP) as their preferred hypothetical service for STI care (women: 58.5% (95% CI 55.2% to 61.6%); men: 54.3% (95% CI 47.1% to 61.3%)). Semistructured interviews elucidated four main responses to symptoms: not seeking healthcare, seeking information to self-diagnose and self-treat, seeking care at non-specialist services and seeking care at sexual health clinics. Collectively, responses suggested individuals sought to gain control over their symptoms, and they prioritised emotional reassurance over accessing medical expertise. Integrating survey and interview data strengthened the evidence that participants preferred their general practitioner for STI care and extended understanding of help-seeking strategies. Conclusions Help-seeking is important to access appropriate healthcare for genitourinary symptoms. Most participants did not attend a sexual health clinic but sought help from other sources. This study supports current service provision options in Britain, facilitating individual autonomy about where to seek help

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