Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins.

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2-like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2-like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.This work was supported by the Wellcome Trust (WT 095195MA). M.D.C. is supported by a BBSRC studentship; L.D. by an EPSRC studentship B.I.M.W. by the Cambridge Trust. JC is a Senior Wellcome Trust Research Fellow.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.R115.69271

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex

A community-based intervention in middle schools to improve HPV vaccination and cervical cancer screening in Japan

Abstract Aim Japan has low rates of cervical cancer screening and Human papilloma virus (HPV) vaccination. This research examines the effectiveness of a family medicine resident-led, intervention in increasing knowledge about HPV and cervical cancer in middle school-girls and increasing knowledge and intention to have cervical cancer screening in their mothers. Methods We utilized a pre-test/post-test intervention design in three rural middle schools with 7th grade middle school-girls and their mothers. A school-based activity educated girls about HPV and cervical cancer. A home-based activity utilized a homework assignment for girls and their mothers. Pre/post intervention surveys were completed by the girls and their mothers. Major outcomes included changes in knowledge among girls and mothers and barriers to be screened for cervical cancer among mothers. Results Sixty-five students and sixty-three mothers completed the study. Two out five mothers were not in compliance with current screening recommendations. Identified barriers included: embarrassment (79%), poor access (56%), fear of having cancer (52%), and cervical cancer screening being an unknown procedure (46%). Forty-four percent of mothers deemed their daughters to be at risk for cervical cancer. Trusted sources of information included: doctors (97%), newspapers/television (89%), government (79%), the Internet (78%), and friends (62%). Student knowledge scores (7-point scale) improved significantly from pre- to post-intervention (4.8 vs. 5.9, p < 0.001). Knowledge scores (14-point scale) among mothers also significantly improved (11.7 vs. 12.0, p = 0.024). Conclusions These data suggest a community-based intervention on a sensitive topic by family medicine residents can be implemented in middle schools, can improve school-girls’ knowledge about HPV and cervical cancer, and can reach their mothers. Additional research could examine whether those intending to be screened receive screening and how to reach women who still resist screening.http://deepblue.lib.umich.edu/bitstream/2027.42/109452/1/12930_2014_Article_13.pd

“Just whack it on until it gets hot”: working with IoT data in the home

This paper presents findings from a co-design project that aims to augment the practices of professional energy advisors with environmental data from sensors deployed in clients’ homes. Premised on prior ethnographic observations we prototyped a sensor platform to support the work of tailoring advice-giving to particular homes. We report on the deployment process and the findings to emerge, particularly the work involved in making sense of or accounting for the data in the course of advice-giving. Our ethnomethodological analysis focuses on the ways in which data is drawn upon as a resource in the home visit, and how understanding and advice-giving turns upon unpacking the indexical relationship of the data to the situated goings-on in the home. This insight, coupled with further design workshops with the advisors, shaped requirements for an interactive system that makes the sensor data available for visual inspection and annotation to support the situated sense-making that is key to giving energy advice

GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements.The US-National Science Foundation and US-National Institutes of Health supported this work through grants MCB-1121867 and 5RO1 NS056114, respectively to R.V.P. J.C. and S.L.S. were supported by the Wellcome Trust (WT 095195MA). M.D.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship.This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/protein/gzw03

“Just whack it on until it gets hot”: working with IoT data in the home

This paper presents findings from a co-design project that aims to augment the practices of professional energy advisors with environmental data from sensors deployed in clients’ homes. Premised on prior ethnographic observations we prototyped a sensor platform to support the work of tailoring advice-giving to particular homes. We report on the deployment process and the findings to emerge, particularly the work involved in making sense of or accounting for the data in the course of advice-giving. Our ethnomethodological analysis focuses on the ways in which data is drawn upon as a resource in the home visit, and how understanding and advice-giving turns upon unpacking the indexical relationship of the data to the situated goings-on in the home. This insight, coupled with further design workshops with the advisors, shaped requirements for an interactive system that makes the sensor data available for visual inspection and annotation to support the situated sense-making that is key to giving energy advice

Thermodynamic mapping of effector protein interfaces with RalA and RalB.

RalA and RalB are members of the Ras family of small G proteins and are activated downstream of Ras via RalGEFs. The RalGEF-Ral axis represents one of the major effector pathways controlled by Ras and as such is an important pharmacological target. RalA and RalB are approximately 80% identical at the amino acid level; despite this, they have distinct roles both in normal cells and in the disease state. We have used our structure of RalB-RLIP76 to guide an analysis of Ral-effector interaction interfaces, creating panels of mutant proteins to probe the energetics of these interactions. The data provide a physical mechanism that underpins the effector selective mutations commonly employed to dissect Ral G protein function. Comparing the energetic landscape of the RalB-RLIP76 and RalB-Sec5 complexes reveals mutations in RalB that lead to differential binding of the two effector proteins. A panel of RLIP76 mutants was used to probe the interaction between RLIP76 and RalA and -B. Despite 100% sequence identity in the RalA and -B contact residues with RLIP76, differences still exist in the energetic profiles of the two complexes. Therefore, we have revealed properties that may account for some of the functional separation observed with RalA and RalB at the cellular level. Our mutations, in both the Ral isoforms and RLIP76, provide new tools that can be employed to parse the complex biology of Ral G protein signaling networks. The combination of these thermodynamic and structural data can also guide efforts to ablate RalA and -B activity with small molecules and peptides.Captain Stephanos FoundationThis is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/bi501530

Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side-effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702 together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses

Lessons learned in developing family medicine residency training programs in Japan

BACKGROUND: While family medicine is not well established as a discipline in Japan, a growing number of Japanese medical schools and training hospitals have recently started sougoushinryoubu (general medicine departments). Some of these departments are incorporating a family medicine approach to residency training. We sought to learn from family medicine pioneers of these programs lessons for developing residency training. METHODS: This qualitative project utilized a long interview research design. Questions focused on four topics: 1) circumstances when becoming chair/faculty member; 2) approach to starting the program; 3) how Western ideas of family medicine were incorporated; and 4) future directions. We analyzed the data using immersion/crystallization to identify recurring themes. From the transcribed data, we selected representative quotations to illustrate them. We verified the findings by emailing the participants and obtaining feedback. RESULTS: Participants included: five chairpersons, two program directors, and three faculty members. We identified five lessons: 1) few people understand the basic concepts of family medicine; 2) developing a core curriculum is difficult; 3) start with undergraduates; 4) emphasize clinical skills; and 5) train in the community. CONCLUSION: While organizational change is difficult, the identified lessons suggest issues that merit consideration when developing a family medicine training program. Lessons from complexity science could inform application of these insights in other countries and settings newly developing residency training