Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 µg/ml or ml · min) and observed 30-40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 µg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 µg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

Escherichia coli Small Proteome.

Escherichia coli was one of the first species to have its genome sequenced and remains one of the best-characterized model organisms. Thus, it is perhaps surprising that recent studies have shown that a substantial number of genes have been overlooked. Genes encoding more than 140 small proteins, defined as those containing 50 or fewer amino acids, have been identified in E. coli in the past 10 years, and there is substantial evidence indicating that many more remain to be discovered. This review covers the methods that have been successful in identifying small proteins and the short open reading frames that encode them. The small proteins that have been functionally characterized to date in this model organism are also discussed. It is hoped that the review, along with the associated databases of known as well as predicted but undetected small proteins, will aid in and provide a roadmap for the continued identification and characterization of these proteins in E. coli as well as other bacteria.

Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes.

Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.IMPORTANCE Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

GroEL actively stimulates folding of the endogenous substrate protein PepQ.

Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

Theorizing the Impact of Targeted Narratives: Model Admiration and Narrative Memorability.

Communication campaigns often include components that have been designed for a specific population, a strategy referred to as targeting. Targeted narratives are story-based components of a campaign that feature a character or situation relevant to the intended audience. Though commonplace, few studies have explicated the underlying mechanisms by which targeted narratives exert influence. In a message evaluation study, 316 women aged 40-75 (Mage = 51.19, SD = 8.11) were exposed to one of two targeted narratives and asked to complete measures of model admiration, narrative memorability, and intentions to receive a mammography. Targeting was based upon affiliation with the Mormon church. The results revealed that the relationship between the targeted narratives and screening intentions was especially strong for women from the target population who admired the depicted models and found the stories memorable.

Colorectal cancer prevention and intentions to use low-dose aspirin: A survey of 1000 U.S. adults aged 40-65.

OBJECTIVE: The Translating Research into Action (TRIA) study was initiated to gather dissemination information on emerging cancer control recommendations. Daily, low-dose aspirin has been identified as a promising means of preventing colorectal cancer, and stakeholders are already calling for research to facilitate dissemination. Thus, the current study sought to identify factors related to intention to use aspirin for colorectal cancer prevention. METHODS: In April 2014, U.S. adults aged 40-65 (N=1000) were recruited to participate in a survey grounded in the health belief model. RESULTS: Older, Black males were more likely to intend to use low-dose aspirin to prevent colorectal cancer. Smokers, and those with a history of polyps, were also more receptive to initiating daily, low-dose aspirin use. Five psychosocial factors were related to intention including self-efficacy, response efficacy, perceived barriers, perceived susceptibility to colorectal cancer, and cancer information overload. CONCLUSION: Initial campaigns/interventions designed to increase daily, low-dose aspirin for colorectal cancer prevention may be more effective if they target receptive populations (older, Black males) using messages informed by the health belief model.

Framing Obesity: How News Frames Shape Attributions and Behavioral Responses.

Based on a public health model of obesity, this study set out to examine whether a news article reporting the obesity issue in a societal versus individual frame would increase perceptions of societal responsibilities for the obesity problem and motivate responsibility-taking behaviors. Responsibility-taking behaviors were examined at 3 levels: personal, interpersonal, and societal. Data from a Web-based experiment revealed significant framing effects on behaviors via causal and treatment responsibility attributions. The societal frame increased societal causal and treatment attribution, which led to greater likelihoods of interpersonal and social responsibility-taking behaviors as well as personal behaviors. Our findings suggest that news framing can be an effective venue for raising awareness of obesity as a societal issue and mobilizing collective efforts.

Explicating perceived barriers to mammography for the USCREEN project: concerns about breast implants, faith violations, and perceived recommendations.

In line with the health belief model, perceived barriers have proven to be a key determinant of intentions to screen for breast cancer. The standard measure of perceived barriers to breast cancer screening is an 11 item scale developed by Victoria Champion. However, perceived barriers emerge and change over time, and Champion's perceived barriers scale was last revised in 1999. Moreover, the original scale did not address barriers which may be more pronounced in particular populations, such as congruity of action with faith. As part of the Utah Screening Project, a sample of women 40-74 (N = 341, Mage = 51.19, SD = 8.11) were recruited from four Utah counties in 2014 to complete a survey. The results revealed that the four new perceived barrier items explained 6.4 % of intentions to screen, above and beyond other predictors. In addition to barriers identified in past research, the current study identified several novel barriers including (a) concerns about negative effects to breast implants, (b) perceived conflict with faith, and the (c) perception that mammography is no longer recommended. The new perceived barriers items are useful to researchers interested in exploring barriers not addressed by the original instrument. The barriers also suggest potential belief-based targets and channels (e.g., plastic surgery clinics, faith-based interventions) for delivering mammography interventions.

Association of Clostridium difficile ribotype 078 with detectable toxin in human stool specimens.

Using a Clostridium difficile glutamate dehydrogenase (GDH) immunoassay and a sensitive C. difficile toxin A/B immunoassay, human stool specimens from patients with diarrhoea (n = 1085) were classified as either GDH positive/toxin negative, or GDH positive/toxin positive. Overall, 528/725 (73%) of the GDH-positive/toxin-negative specimens contained viable C. difficile, and 433/528 (82%) of these C. difficile isolates were PCR positive for the toxin gene pathogenicity locus. Overall, 867/1078 (80%) of the GDH-positive specimens contained viable C. difficile, and 433/725 (60%) of the GDH-positive/toxin-negative specimens contained a toxigenic C. difficile strain. The diversity of toxigenic C. difficile ribotypes isolated from toxin-negative specimens (n = 433) and toxin-positive specimens (n = 339) was significantly different (P < 0.0001). Specifically, the presence of ribotype 078 strains was very strongly associated (P < 0.0001) with detection of toxin in clinical specimens using a sensitive toxin immunoassay. Specimens positive for ribotype 078 were almost twice as likely to be toxin positive as opposed to toxin negative (risk ratio = 1.90, 95% confidence interval 1.64-2.19). In contrast, other circulating ribotypes were seen with similar frequency in specimens with and without detectable toxin. This supports the view that ribotype 078 strains may be more virulent than other common ribotypes in terms of toxin production.

Psychosocial predictors of human papillomavirus vaccination intentions for young women 18 to 26: religiosity, morality, promiscuity, and cancer worry.

OBJECTIVES: To determine whether five psychosocial variables, namely, religiosity, morality, perceived promiscuity, cancer worry frequency, and cancer worry severity, predict young women's intentions to receive the human papillomavirus (HPV) vaccination. METHODS: Female undergraduate students (n=408) completed an online survey. Questions pertaining to hypothesized predictors were analyzed through bivariate correlations and hierarchical regression equations. Regressions examined whether the five psychosocial variables of interest predicted intentions to vaccinate above and beyond controls. Proposed interactions among predictor variables were also tested. RESULTS: Study findings supported cancer worry as a direct predictor of HPV vaccination intention, and religiosity and sexual experience as moderators of the relationship between concerns of promiscuity reputation and intentions to vaccinate. One dimension of cancer worry (severity) emerged as a particularly robust predictor for this population. CONCLUSIONS: This study provides support for several important, yet understudied, factors contributing to HPV vaccination intentions among college-aged women: cancer worry severity and religiosity. Future research should continue to assess the predictive contributions of these variables and evaluate how messages and campaigns to increase HPV vaccination uptake can utilize religious involvement and worry about cancer to promote more effectively HPV vaccination as a cancer prevention strategy.

The Health Belief Model as an explanatory framework in communication research: exploring parallel, serial, and moderated mediation.

The Health Belief Model (HBM) posits that messages will achieve optimal behavior change if they successfully target perceived barriers, benefits, self-efficacy, and threat. While the model seems to be an ideal explanatory framework for communication research, theoretical limitations have limited its use in the field. Notably, variable ordering is currently undefined in the HBM. Thus, it is unclear whether constructs mediate relationships comparably (parallel mediation), in sequence (serial mediation), or in tandem with a moderator (moderated mediation). To investigate variable ordering, adults (N = 1,377) completed a survey in the aftermath of an 8-month flu vaccine campaign grounded in the HBM. Exposure to the campaign was positively related to vaccination behavior. Statistical evaluation supported a model where the indirect effect of exposure on behavior through perceived barriers and threat was moderated by self-efficacy (moderated mediation). Perceived barriers and benefits also formed a serial mediation chain. The results indicate that variable ordering in the Health Belief Model may be complex, may help to explain conflicting results of the past, and may be a good focus for future research.

Structural basis of substrate selectivity of E. coli prolidase.

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

The C-terminal tails of the bacterial chaperonin GroEL stimulate protein folding by directly altering the conformation of a substrate protein.

Many essential cellular proteins fold only with the assistance of chaperonin machines like the GroEL-GroES system of Escherichia coli. However, the mechanistic details of assisted protein folding by GroEL-GroES remain the subject of ongoing debate. We previously demonstrated that GroEL-GroES enhances the productive folding of a kinetically trapped substrate protein through unfolding, where both binding energy and the energy of ATP hydrolysis are used to disrupt the inhibitory misfolded states. Here, we show that the intrinsically disordered yet highly conserved C-terminal sequence of the GroEL subunits directly contributes to substrate protein unfolding. Interactions between the C terminus and the non-native substrate protein alter the binding position of the substrate protein on the GroEL apical surface. The C-terminal tails also impact the conformational state of the substrate protein during capture and encapsulation on the GroEL ring. Importantly, removal of the C termini results in slower overall folding, reducing the fraction of the substrate protein that commits quickly to a productive folding pathway and slowing several kinetically distinct folding transitions that occur inside the GroEL-GroES cavity. The conserved C-terminal tails of GroEL are thus important for protein folding from the beginning to the end of the chaperonin reaction cycle.

Synthetic inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery.

Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.

Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response.

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

Understanding inositol pyrophosphate metabolism and function: kinetic characterization of the DIPPs.

We illuminate the metabolism and the cell-signaling activities of inositol pyrophosphates, by showing that regulation of yeast cyclin-kinase by 1-InsP7 is not conserved for mammalian CDK5, and by kinetically characterizing Ddp1p/DIPP-mediated dephosphorylation of 1-InsP7, 5-InsP7 and InsP8. Each phosphatase exhibited similar Km values for every substrate (range: 35-148 nM). The rank order of kcat values (1-InsP7>5-InsP7=InsP8) was identical for each enzyme, although DIPP1 was 10- to 60-fold more active than DIPP2α/ß and DIPP3α/ß. We demonstrate InsP8 dephosphorylation preferentially progresses through 1-InsP7. Conversely, we conclude that the more metabolically and functionally significant steady-state route of InsP8 synthesis proceeds via 5-InsP7.

Visualizing GroEL/ES in the act of encapsulating a folding protein.

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains.

We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 µM and InsP6=7 µM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 µM and 1-InsP7=43 µM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.

Functional analysis of a class I holin, P2 Y.

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character.