X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His. Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC. The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A. From the structures, we were able to delineate alternate proton transfer pathways in the revertants. The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213. The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes

An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities. Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis [Kaser et al., Biol. Chem. 382 (2001) 1637-1647]. Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast. Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival. We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains. In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit. This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus. Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene.     

Substituted 2-(R)-methyl piperazines as muscarinic M(2) selective ligands

A novel series of 2-(R)-methyl-substituted piperazines (e.g., 2) is described. They are potent M(2) selective ligands that have >100-fold selectivity versus the M(1) receptor. In the rat microdialysis assay, compound 14 showed significantly enchanced levels of acetylcholine after oral administration.     

The Personality Behind Cheating: Behavioural Types and the Feeding Ecology of Cleaner Fish

The complex mutualistic relationship between the cleaner fish (Labroides dimidiatus) and their ‘clients’ in many reef systems throughout the world has been the subject of debate and research interest for decades. Game‐theory models have long struggled with explaining how the mixed strategies of cheating and honesty might have evolved in such a system and while significant efforts have been made theoretically, demonstrating the nature of this relationship empirically remains an important research challenge. Using the experimental framework of behavioural syndromes, we sought to quantitatively assess the relationship between personality and the feeding ecology of cleaner fish to provide novel insights into the underlying mechanistic basis of cheating in cleaner‐client interactions. First, we observed and filmed cleaner fish interactions with heterospecifics, movement patterns and general feeding ecology in the wild. We then captured and measured all focal individuals and tested them for individual consistency in measures of activity, exploration and risk taking (boldness) in the laboratory. Our results suggest a syndrome incorporating aspects of personality and foraging effort are central components of the behavioural ecology of L. dimidiatus on the Great Barrier Reef. We found that individuals that exhibited greater feeding effort tended to cheat proportionately less and move over smaller distances relative to bolder more active, exploratory individuals. Our study demonstrates for the first time that individual differences in personality might be mechanistically involved in explaining how the mixed strategies of cheating and honesty persist in cleaner fish mutualisms.     

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild‐type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non‐photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild‐type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light‐regulated manner.     

Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.Glucagon-like peptide-1 (GLP-1)¹ is a gastrointestinal hormone secreted by intestinal L cells upon food intake that is best known for its role in controlling glucose homeostasis. Acting through its cognate glucagon-like peptide-1 receptor (GLP-1R), GLP-1 has several important physiological and pharmacological functions. GLP-1 is best known for enhancing glucose-stimulated insulin secretion (GSIS) from the pancreatic β cells. Importantly, the insulinotropic properties of GLP-1 are maintained in patients with type 2 diabetes (1), which is characterized by insufficient insulin secretion from pancreatic β cells and an inability to maintain glucose homeostasis. Therefore, therapeutic strategies targeting GLP-1R have been developed to treat type 2 diabetes (2, 3). In addition to augmenting insulin secretion, GLP-1 has been known to improve glucose sensing, proinsulin biosynthesis, survival, and proliferation of β cells (3, 4) in a variety of experimental models. GLP-1 also has several extrapancreatic effects, including actions on the central nervous system to inhibit food intake (5), the stomach to decrease gastric emptying and gastric acid secretion (6), and the lungs to stimulate secretion of macromolecules from airways (7). Additionally, GLP-1 has an effect on the heart and possibly the kidney to modulate blood pressure and heart rate (8, 9).The GLP-1R is a member of the B1 family of G protein–coupled receptors (secretin receptor family). In mammals, GLP-1R is expressed in multiple tissues, including pancreatic β cells and δ cells (10), hypothalamus, lung, stomach, heart, kidney (11), and thyroid (12), which in part explains its diverse actions. Upon ligand binding, the GLP-1R is capable of coupling to diverse cell signal transduction pathways, but it is best known for its actions on G protein Gs α and adenylate cyclase activity to increase intracellular cAMP. It is known that other proteins can affect GLP-1R activity in addition to G proteins, including β-arrestin and caveolin, which affect receptor internalization and trafficking. β-Arrestin 1 is also required for proper GLP-1-stimulated cAMP production (13–15). More recently, it was shown that another B1 family member, gastric inhibitory polypeptide receptor heterodimerizes with GLP-1R, decreasing GLP-1-induced β-arrestin recruitment and mobilization (16). Very recently, our group identified several novel potential GLP-1R interactors using a membrane-based split-ubiquitin yeast two-hybrid (MYTH) assay (17). Three β cell–expressing membrane-bound interactors, solute carrier family 15 member 4 (SLC15A4), amyloid β A4 precursor-like protein 1 (APLP1), and adaptor-related protein complex 2 subunit mu (AP2M1), were further selected for individual knockdown in mouse insulinoma (MIN6) β cells using small interfering RNAs (siRNAs). GLP-1-induced insulin secretion was significantly enhanced when these genes were silenced, suggesting that these interactor proteins attenuate GLP-1R activity. These findings demonstrated that GLP-1R protein interactions are complex and the interactors can have measurable effects on receptor trafficking and downstream signaling. Such interactions may in part explain the diverse tissue-specific effects of GLP-1 and offer avenues for controlling GLP-1 actions in a tissue-selective manner.Although the MYTH system is well established (18) and has been applied to study G protein–coupled receptor interactomes (17), it is limited on two fronts. Firstly, it must be performed in yeast which is not an ideal representation of the mammalian system. Secondly, it is technically difficult to activate the receptor in MYTH, thus, effects of ligand stimulation on the receptor interactome cannot be assessed. Recently, affinity purification–mass spectrometry (AP-MS) has become a powerful tool for discovering and examining novel protein–protein interactions, including those between membrane-bound proteins in mammalian cells (19–21). In the current study, we applied AP-MS to discover novel GLP-1R interactors and employed a human GLP-1R harboring a FLAG^® epitope. GLP-1R-Flag was expressed in either Chinese hamster ovary (CHO) cells or MIN6 β cells, and interactors were studied in the presence or absence of GLP-1.     

Generation of “Virtual” Control Groups for Single Arm Prostate Cancer Adjuvant Trials

It is difficult to construct a control group for trials of adjuvant therapy (Rx) of prostate cancer after radical prostatectomy (RP) due to ethical issues and patient acceptance. We utilized 8 curve-fitting models to estimate the time to 60%, 65%, … 95% chance of progression free survival (PFS) based on the data derived from Kattan post-RP nomogram. The 8 models were systematically applied to a training set of 153 post-RP cases without adjuvant Rx to develop 8 subsets of cases (reference case sets) whose observed PFS times were most accurately predicted by each model. To prepare a virtual control group for a single-arm adjuvant Rx trial, we first select the optimal model for the trial cases based on the minimum weighted Euclidean distance between the trial case set and the reference case set in terms of clinical features, and then compare the virtual PFS times calculated by the optimum model with the observed PFSs of the trial cases by the logrank test. The method was validated using an independent dataset of 155 post-RP patients without adjuvant Rx. We then applied the method to patients on a Phase II trial of adjuvant chemo-hormonal Rx post RP, which indicated that the adjuvant Rx is highly effective in prolonging PFS after RP in patients at high risk for prostate cancer recurrence. The method can accurately generate control groups for single-arm, post-RP adjuvant Rx trials for prostate cancer, facilitating development of new therapeutic strategies.