Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Structure of the p53 degradation complex from HPV16.

Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders.

Protein-protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions with these topologies and advance the development of PPI-focused therapeutics, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand and demonstrate its application in a model PPI. Cysteine mutants of a known ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a model PPI with the 9-mer peptide antigen VL9 and major histocompatibility complex (MHC) class I protein HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein-peptide conjugate within 4 h. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94-NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential application of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Discovery of reactive peptide inhibitors of human papillomavirus oncoprotein E6.

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

Cryo-EM structure of human PAPP-A2 and mechanism of substrate recognition.

Pregnancy-Associated Plasma Protein A isoforms, PAPP-A and PAPP-A2, are metalloproteases that cleave insulin-like growth factor binding proteins (IGFBPs) to modulate insulin-like growth factor signaling. The structures of homodimeric PAPP-A in complex with IGFBP5 anchor peptide, and inhibitor proteins STC2 and proMBP have been recently reported. Here, we present the single-particle cryo-EM structure of the monomeric, N-terminal LG, MP, and the M1 domains (with the exception of LNR1/2) of human PAPP-A2 to 3.13 Å resolution. Our structure together with functional studies provides insight into a previously reported patient mutation that inactivates PAPP-A2 in a distal region of the protein. Using a combinational approach, we suggest that PAPP-A2 recognizes IGFBP5 in a similar manner as PAPP-A and show that PAPP-A2 cleaves IGFBP5 less efficiently due to differences in the M2 domain. Overall, our studies characterize the cleavage mechanism of IGFBP5 by PAPP-A2 and shed light onto key differences with its paralog PAPP-A.

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein.

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

Pathological gaming: a longitudinal study from the perspectives of mental health problems and social stress model.

Debates about pathological gaming continues in the wake of the World Health Organization's (WHO) decision to establish a gaming disorder diagnosis. Questions persist whether gaming disorder is best conceived as a stand-alone psychiatric disorder, or whether it heralds or accompanies other, more established conditions, such as depression or ADHD. We tested these hypotheses in a sample of 3,034 youth from Singapore. Evidence suggests that pathological gaming is a somewhat unstable construct, often remitting spontaneously. Youth with preexisting ADHD or depression were more likely to develop later pathological gaming problems, while the inverse was not true, with neither early pathological gaming nor gaming time predictive of later mental health problems. Results suggest that, whenever there is any need to conduct robust evidence-based studies, more evidence should be collected before new disorders are recognized by means of "expert consensus".

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition.

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

Triple-threat quantitative multiplexed plasma proteomics analysis on immune complex disease MRL-lpr mice.

Systemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes. Methodologies used included immunohistochemistry, immunoglobulin isotyping, multiplexed proteome profiling, immunoglobulin immunoprecipitation with glycoproteome profiling, and size exclusion chromatography (SEC) profiling to enable a comprehensive proteome profiling of proteins and protein complexes. We also used a novel native multiplexed plasma proteome profiling (NativeMP3) method that relies on native enrichment of plasma proteins enabling ultra-deep single shot profiling where we identified 922 plasma proteins at 1% false discovery rate (FDR) in a single shot mass spectrometry run. We observed many large plasma protein differences between the MRL-lpr and control strain including differences in the immunoglobulins, immunoglobulins against specific antigens, chemokines, and proteases as well as changes in protein complexes such as the immunoproteasome.

Aggressive Video Games are Not a Risk Factor for Future Aggression in Youth: A Longitudinal Study.

The issue of whether video games with aggressive or violent content (henceforth aggressive video games) contribute to aggressive behavior in youth remains an issue of significant debate. One issue that has been raised is that some studies may inadvertently inflate effect sizes by use of questionable researcher practices and unstandardized assessments of predictors and outcomes, or lack of proper theory-driven controls. In the current article, a large sample of 3034 youth (72.8% male Mage = 11.2) in Singapore were assessed for links between aggressive game play and seven aggression or prosocial outcomes 2 years later. Theoretically relevant controls for prior aggression, poor impulse control, gender and family involvement were used. Effect sizes were compared to six nonsense outcomes specifically chosen to be theoretically unrelated to aggressive game play. The use of nonsense outcomes allows for a comparison of effect sizes between theoretically relevant and irrelevant outcomes, to help assess whether any statistically significant outcomes may be spurious in large datasets. Preregistration was employed to reduce questionable researcher practices. Results indicate that aggressive video games were unrelated to any of the outcomes using the study criteria for significance. It would take 27 h/day of M-rated game play to produce clinically noticeable changes in aggression. Effect sizes for aggression/prosocial outcomes were little different than for nonsense outcomes. Evidence from this study does not support the conclusion that aggressive video games are a predictor of later aggression or reduced prosocial behavior in youth.

Motivations for Volunteering and Its Associations with Time Perspectives and Life Satisfaction: A Latent Profile Approach.

This study aims to examine motivation for volunteering and its association with time perspective and life satisfaction among volunteers ( N = 221). Latent profile analysis was used to profile individuals based on their time perspectives and then to compare group differences in life satisfaction and volunteering motivation. Three profiles were identified. Profile 1 ( n = 32; 14.5%) was a "balanced time perspective group," Profile 2 ( n = 102; 46.2%) was a "maladaptive group," and Profile 3 ( n = 87; 39.3%) was a "nonchalant group." Profile 1 showed the highest life satisfaction compared to the two remaining groups. Significant group differences in volunteering motivation between this group and the other two were also reported. These findings suggest that time perspective may be appropriate for understanding motivation for voluntarism and life satisfaction.

Students' Motivational Profiles in the Physical Education Context.

The purpose of the current study is to test the self-determination theory (SDT) continuum hypothesis of motivation using latent profile analysis (LPA). A total of 3,220 school students took part in the study. We compared LPA solutions estimated using the four motivation types versus the two higher-order dimensions to assess their degree of correspondence to the SDT continuum hypothesis. To examine the concurrent validity of the profiles, we also verified their associations with three predictors (age, gender, perception of physical education teachers' autonomy-supportive behaviors) and two outcomes variables (perceived competence and intentions to be physically active). The results showed that profiling using the four motivation types provides more differentiated and meaningful description of responses to the Perceived Locus of Causality Scale, compared with profiling using two higher-order factors. In general, the results of the current study were consistent with the SDT continuum hypothesis of human motivation.

Chronic inhibition, self-control and eating behavior: test of a 'resource depletion' model.

The current research tested the hypothesis that individuals engaged in long-term efforts to limit food intake (e.g., individuals with high eating restraint) would have reduced capacity to regulate eating when self-control resources are limited. In the current research, body mass index (BMI) was used as a proxy for eating restraint based on the assumption that individuals with high BMI would have elevated levels of chronic eating restraint. A preliminary study (Study 1) aimed to provide evidence for the assumed relationship between eating restraint and BMI. Participants (N = 72) categorized into high or normal-range BMI groups completed the eating restraint scale. Consistent with the hypothesis, results revealed significantly higher scores on the weight fluctuation and concern for dieting subscales of the restraint scale among participants in the high BMI group compared to the normal-range BMI group. The main study (Study 2) aimed to test the hypothesized interactive effect of BMI and diminished self-control resources on eating behavior. Participants (N = 83) classified as having high or normal-range BMI were randomly allocated to receive a challenging counting task that depleted self-control resources (ego-depletion condition) or a non-depleting control task (no depletion condition). Participants then engaged in a second task in which required tasting and rating tempting cookies and candies. Amount of food consumed during the taste-and-rate task constituted the behavioral dependent measure. Regression analyses revealed a significant interaction effect of these variables on amount of food eaten in the taste-and-rate task. Individuals with high BMI had reduced capacity to regulate eating under conditions of self-control resource depletion as predicted. The interactive effects of BMI and self-control resource depletion on eating behavior were independent of trait self-control. Results extend knowledge of the role of self-control in regulating eating behavior and provide support for a limited-resource model of self-control.

Development of the talent development environment questionnaire for sport.

As sporting challenge at the elite level becomes ever harder, maximizing effectiveness of the talent development pathway is crucial. Reflecting this need, this paper describes the development of the Talent Development Environment Questionnaire, which has been designed to facilitate the development of sporting potential to world-class standard. The questionnaire measures the experiences of developing athletes in relation to empirically identified "key features" of effective talent development environments. The first phase involved the generation of questionnaire items with clear content and face validity. The second phase explored the factor structure and reliability. This was carried out with 590 developing athletes through application of exploratory factor analysis with oblique rotation, principal axis factoring extraction and cronbach alpha tests. This yielded a 59-item, seven-factor structure with good internal consistency (0.616-0.978). The Talent Development Environment Questionnaire appears to be a promising psychometric instrument that can potentially be useful for education and formative review in applied settings, and as a measurement tool in talent development research.

Evaluating the effects of implementation intention and self-concordance on behaviour.

The purpose of the present study was to investigate effects of implementation intentions on taking one multivitamin tablet, everyday, for 2 weeks, among individuals who endorsed self-concordant and self-discordant forms of motivation. A 2 (implementation intentions: yes, no) × 3 (motivation: self-concordance, self-discordance, control) experimental design was adopted with university students being exposed to manipulations of implementation intentions, self-concordance, and self-discordance (male = 110, female = 120, M age = 23.50 years, SD = 7.21). Results of the study indicated that while implementation intentions increased multivitamin intake for individuals who endorsed self-concordant and self-discordant forms of motivation, the combination of self-concordance and implementation intentions produced particularly enhanced levels of compliance on multivitamin intake. The implications of results of the present study to theory development and practice are discussed.

Effects of a 12-week exercise training programme on aerobic fitness, body composition, blood lipids and C-reactive protein in adolescents with obesity.

INTRODUCTION: Developing effective exercise programmes for the paediatric population is a strategy for decreasing obesity and is expected to help in eventually limiting obesity-associated long-term health and societal impact. In this study, the effects of a 12-week twice weekly additional exercise training, which comprised a combination of circuit-based resistance training and aerobic exercises, in additional to typical physical education sessions, on aerobic fitness, body composition and serum C-reactive protein (CRP) and lipids were analysed in 13- to 14-year-old obese boys contrasted with a control group. MATERIALS AND METHODS: Both the exercise group (EG, n = 12) and control group (CG, n = 12) participated in the typical 2 sessions of 40-minute physical education (PE) per week in schools, but only EG participated in additional 2 sessions per week of 45 to 60 minutes per session of exercise training, which comprised a combination of circuit-based resistance training and aerobic exercises maintained at 65% to 85% maximum heart rate (HRmax = 220 - age). Body composition was measured using dual energy X-ray absorptiometry (DEXA). Fasting serum CRP and blood lipids were analysed pre- and postexercise programme. Aerobic fitness was measured by an objective laboratory submaximal exercise test, PWC170 (Predicted Work Capacity at HR 170 bpm). RESULTS: Exercise training significantly improved lean muscle mass, body mass index, fitness, resting HR, systolic blood pressure and triglycerides in EG. Serum CRP concentrations were elevated at baseline in both groups, but training did not result in a change in CRP levels. In the CG, body weight increased significantly at the end of the 12-week period. CONCLUSION: This study supports the value of an additional exercise training programme, beyond the typical twice weekly physical education classes, to produce physiological benefits in the management of obesity in adolescents, including prevention of weight gain.

Cross-cultural generalizability of the theory of planned behavior among young people in a physical activity context.

The present study tested the cross-cultural generalizability of the measurement and structural parameters of the theory of planned behavior (TPB) among youth in a physical activity context. Pupils from five cultural groups completed measures of attitudes, subjective norms, perceived behavioral control (PBC), and intentions for physical activity. Five weeks later, participants completed self-report measures of physical activity behavior. Confirmatory factor analyses and multisample structural equation models revealed well-fitting models within each sample with minimal variations in the measurement parameters across cultures. There were a few significant cross-cultural differences in the structural relations among the TPB constructs. Attitudes predicted intentions in all samples (beta range = .300 to .550), whereas the effect of the subjective norms on intention was nonsignificant in all but the Hungarian sample (beta = .243). Conversely, the effect of PBC on intentions was significant (beta range = .302 to .573) in all but the Hungarian sample. Findings support the generalizability of the measures and pattern of effects for the TPB among young people in a physical activity context.