Publications by authors named "Dusan Petrovic"

41 Publications

Investigating the Relations Between Caffeine-Derived Metabolites and Plasma Lipids in 2 Population-Based Studies.

Mayo Clin Proc 2021 Sep 25. Epub 2021 Sep 25.

Department and Division of Primary Care Medicine, Geneva University Hospitals (HUG), Switzerland. Electronic address:

Objective: To investigate the relations between caffeine-derived metabolites (methylxanthines) and plasma lipids by use of population-based data from 2 European countries.

Methods: Families were randomly selected from the general population of northern Belgium (FLEMENGHO), from August 12, 1985, until November 22, 1990, and 3 Swiss cities (SKIPOGH), from November 25, 2009, through April 4, 2013. We measured plasma concentrations (FLEMENGHO, SKIPOGH) and 24-hour urinary excretions (SKIPOGH) of 4 methylxanthines-caffeine, paraxanthine, theobromine, and theophylline-using ultra-high-performance liquid chromatography-tandem mass spectrometry. We used enzymatic methods to estimate total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels and the Friedewald equation for low-density lipoprotein cholesterol levels in plasma. We applied sex-specific mixed models to investigate associations between methylxanthines and plasma lipids, adjusting for major confounders.

Results: In both FLEMENGHO (N=1987; 1055 [53%] female participants) and SKIPOGH (N=990; 523 [53%] female participants), total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels increased across quartiles of plasma caffeine, paraxanthine, and theophylline (total cholesterol levels by caffeine quartiles in FLEMENGHO, male participants: 5.01±0.06 mmol/L, 5.05±0.06 mmol/L, 5.27±0.06 mmol/L, 5.62±0.06 mmol/L; female participants: 5.24±0.06 mmol/L, 5.15±0.05 mmol/L, 5.25±0.05 mmol/L, 5.42±0.05 mmol/L). Similar results were observed using urinary methylxanthines in SKIPOGH (total cholesterol levels by caffeine quartiles, male participants: 4.54±0.08 mmol/L, 4.94±0.08 mmol/L, 4.87±0.08 mmol/L, 5.27±0.09 mmol/L; female participants: 5.12±0.07 mmol/L, 5.21±0.07 mmol/L, 5.28±0.05 mmol/L, 5.28±0.07 mmol/L). Furthermore, urinary caffeine and theophylline were positively associated with high-density lipoprotein cholesterol in SKIPOGH male participants.

Conclusion: Plasma and urinary caffeine, paraxanthine, and theophylline were positively associated with plasma lipids, whereas the associations involving theobromine were less clear. We postulate that the positive association between caffeine intake and plasma lipids may be related to the sympathomimetic function of methylxanthines, mitigating the overall health-beneficial effect of caffeine intake.
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http://dx.doi.org/10.1016/j.mayocp.2021.05.030DOI Listing
September 2021

Health Insurance Deductibles and Health Care-Seeking Behaviors in a Consumer-Driven Health Care System With Universal Coverage.

JAMA Netw Open 2021 Jul 1;4(7):e2115722. Epub 2021 Jul 1.

Unit of Population Epidemiology, Division and Department of Primary Care Medicine, Geneva University Hospitals, Geneva, Switzerland.

Importance: Characteristics of a health care system can facilitate forgoing of health care owing to economic reasons and can influence population health. Whether health insurance deductibles are associated with forgoing of health care in a consumer-driven health care system with universal coverage, such as the Swiss health system, remains to be determined.

Objective: To assess the association between insurance plan deductibles and forgoing of health care with consideration of socioeconomic factors.

Design, Setting, And Participants: This cross-sectional study was conducted in Geneva, Switzerland, using data collected from January 1, 2007, to December 31, 2019. Population-based samples were obtained yearly through random stratified sampling by age and sex of the general population aged 20 to 74 years. Participants were invited to an appointment at 1 of the 3 study sites in Geneva, where they completed a sociodemographic and health questionnaire.

Exposures: Insurance plan deductible level.

Main Outcomes And Measures: The main outcome was forgoing of health care owing to economic reasons. Unadjusted and multivariable Poisson models were used to assess the association between deductible level and forgoing of health care. Differences in forgoing health care across the range of health insurance deductibles or household income levels were quantified using the relative index of inequality (RII).

Results: The study group included 11 872 participants (5974 [50.3%] male; median age, 48.1 years [interquartile range, 38.7-59.1 years]); 1146 (9.7%) reported forgoing health care. Participants with high-deductible plans reported forgoing health care more frequently than those with low-deductible plans (331 [13.5%] vs 591 [8.7%]). In adjusted analysis, higher-deductible plans were associated with a greater likelihood of forgoing health care (RII, 2.2; 95% CI, 1.7-3.0; P < .001) independently of socioeconomic status, known comorbidities, and cardiovascular risk factors. Deductible level was associated with forgoing of health care among participants younger than 40 years (RII, 2.5; 95% CI, 1.6-4.0; P < .001) and those aged 40 to 64 years (RII, 1.9; 95% CI, 1.3-2.9; P = .002) but not among those older than 65 years (RII, 2.9; 95% CI, 0.8-10.4; P = .11).

Conclusions And Relevance: In this cross-sectional study, high insurance plan deductibles were associated with forgoing of health care independent of socioeconomic status and preexisting conditions in a universal consumer-driven health care system with good population outcomes in Switzerland. Uncovering health care system design features that could lead to suboptimal population care may help decision makers improve their current health care system design to achieve better outcomes.
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http://dx.doi.org/10.1001/jamanetworkopen.2021.15722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261614PMC
July 2021

Insights into household transmission of SARS-CoV-2 from a population-based serological survey.

Nat Commun 2021 06 15;12(1):3643. Epub 2021 Jun 15.

Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.

Understanding the risk of infection from household- and community-exposures and the transmissibility of asymptomatic infections is critical to SARS-CoV-2 control. Limited previous evidence is based primarily on virologic testing, which disproportionately misses mild and asymptomatic infections. Serologic measures are more likely to capture all previously infected individuals. We apply household transmission models to data from a cross-sectional, household-based population serosurvey of 4,534 people ≥5 years from 2,267 households enrolled April-June 2020 in Geneva, Switzerland. We found that the risk of infection from exposure to a single infected household member aged ≥5 years (17.3%,13.7-21.7) was more than three-times that of extra-household exposures over the first pandemic wave (5.1%,4.5-5.8). Young children had a lower risk of infection from household members. Working-age adults had the highest extra-household infection risk. Seropositive asymptomatic household members had 69.4% lower odds (95%CrI,31.8-88.8%) of infecting another household member compared to those reporting symptoms, accounting for 14.5% (95%CrI, 7.2-22.7%) of all household infections.
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http://dx.doi.org/10.1038/s41467-021-23733-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206123PMC
June 2021

Prospective Identification of Elevated Circulating CDCP1 in Patients Years before Onset of Lung Cancer.

Cancer Res 2021 Jul 11;81(13):3738-3748. Epub 2021 Feb 11.

MRC Centre for Environment and Health, Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, United Kingdom.

Increasing evidence points to a role for inflammation in lung carcinogenesis. A small number of circulating inflammatory proteins have been identified as showing elevated levels prior to lung cancer diagnosis, indicating the potential for prospective circulating protein concentration as a marker of early carcinogenesis. To identify novel markers of lung cancer risk, we measured a panel of 92 circulating inflammatory proteins in 648 prediagnostic blood samples from two prospective cohorts in Italy and Norway (women only). To preserve the comparability of results and protect against confounding factors, the main statistical analyses were conducted in women from both studies, with replication sought in men (Italian participants). Univariate and penalized regression models revealed for the first time higher blood levels of CDCP1 protein in cases that went on to develop lung cancer compared with controls, irrespective of time to diagnosis, smoking habits, and gender. This association was validated in an additional 450 samples. Associations were stronger for future cases of adenocarcinoma where CDCP1 showed better explanatory performance. Integrative analyses combining gene expression and protein levels of CDCP1 measured in the same individuals suggested a link between CDCP1 and the expression of transcripts of LRRN3 and SEM1. Enrichment analyses indicated a potential role for CDCP1 in pathways related to cell adhesion and mobility, such as the WNT/β-catenin pathway. Overall, this study identifies lung cancer-related dysregulation of CDCP1 expression years before diagnosis. SIGNIFICANCE: Prospective proteomics analyses reveal an association between increased levels of circulating CDCP1 and lung carcinogenesis irrespective of smoking and years before diagnosis, and integrating gene expression indicates potential underlying mechanisms..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-3454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611235PMC
July 2021

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase.

Nat Commun 2021 01 15;12(1):380. Epub 2021 Jan 15.

Departamento de Quimica Fisica. Facultad de Ciencias, Unidad de Excelencia de Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Universidad de Granada, 18071, Granada, Spain.

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.
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http://dx.doi.org/10.1038/s41467-020-20630-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810902PMC
January 2021

Education, biological ageing, all-cause and cause-specific mortality and morbidity: UK biobank cohort study.

EClinicalMedicine 2020 Dec 19;29-30:100658. Epub 2020 Nov 19.

Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, Netherlands.

Background: Socioeconomic position as measured by education may be embodied and affect the functioning of key physiological systems. Links between social disadvantage, its biological imprint, and cause-specific mortality and morbidity have not been investigated in large populations, and yet may point towards areas for public health interventions beyond targeting individual behaviours.

Methods: Using data from 366,748 UK Biobank participants with 13 biomarker measurements, we calculated a Biological Health Score (BHS, ranging from 0 to 1) capturing the level of functioning of five physiological systems. Associations between BHS and incidence of cardiovascular disease (CVD) and cancer, and mortality from all, CVD, cancer, and external causes were examined. We explored the role of education in these associations. Mendelian randomisation using genetic evidence was used to triangulate these findings.

Findings: An increase in BHS of 0.1 was associated with all-cause (HR = 1.14 [1.12-1.16] and 1.09 [1.07-1.12] in men and women respectively), cancer (HR = 1.11 [1.09-1.14] and 1.07 [1.04-1.10]) and CVD (HR = 1.25 [1.20-1.31] and 1.21 [1.11-1.31]) mortality, CVD incidence (HR = 1.15 [1.13-1.16] and 1.17 [1.15-1.19]). These associations survived adjustment for education, lifestyle-behaviours, body mass index (BMI), co-morbidities and medical treatments. Mendelian randomisation further supported the link between the BHS and CVD incidence (HR = 1.31 [1.21-1.42]). The BHS contributed to CVD incidence prediction (age-adjusted C-statistic = 0.58), other than through education and health behaviours.

Interpretation: The BHS captures features of the embodiment of education, health behaviours, and more proximal unknown factors which all complementarily contribute to all-cause, cancer and CVD morbidity and premature death.
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http://dx.doi.org/10.1016/j.eclinm.2020.100658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788440PMC
December 2020

Impact of obesity with or without hypertension on systemic haemodynamic and renal responses to lower body negative pressure.

Blood Press 2021 02 19;30(1):67-74. Epub 2020 Oct 19.

Service of Nephrology and Hypertension, Lausanne University Hospital (CHUV) and University of Lausanne, Switzerland.

Purpose: Sodium and water handling by the kidney and the sympathetic nervous system have been implicated in the development of obesity-related hypertension and kidney disease. They have seldom been studied together during stress conditions. The objective of this study was to compare the systemic, renal and hormonal responses to lower body negative pressure (LBNP) in adult healthy participants (H), obese normotensive (OBN) and obese hypertensive patients (OBH).

Materials And Methods: This was a prospective case-control study. Participants from the three groups were exposed to one hour of LBNP. Systemic and renal haemodynamics, sodium and water excretion and hormones were measured before and after LBNP. Intergroup LBNP responses were tested using a Student t-test or a Wilcoxon rank-sum test. An extension of the Wilcoxon rank-sum test was used to test for a trend across the three groups.

Results: The study included 54 participants (H: 25, OBN: 16, OBH: 13). LBNP induced a stepwise increase in systolic blood pressure (+2.7 ± 4.7 mmHg (H) +4.7 ± 8.8 mmHg (OBN) +8.0 ± 8.6 mmHg (OBH,  = .028)) and heart rate (-1.3 ± 4.9 bpm (H) 2.2 ± 6.1 bpm (OBN) 1.9 ± 4.1 bpm (OBH,  = .041). Urinary output (-2.8 ± 2.1 ml/min -1.4 ± 1.7 ml/min,  = .028) and free water clearance (-1.9 ± 1.7 mOsm/kg -0.7 ± 1.3 mOsm/kg,  = .016) responses were more marked in OBN compared to H.

Conclusions: These results show that the systemic and the renal response to LBNP differ according to weight and to BP categories. Systolic BP and heart show a progressive increased response form healthy volunteers to OBN and then to obese hypertensive participants while urinary output and free water clearance responses are increased in OBN only, suggesting that the occurrence of hypertension in obese individuals modifies the early kidney responses to stress.

Clinicaltrial.gov Identifier: NCT01734096.
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http://dx.doi.org/10.1080/08037051.2020.1829963DOI Listing
February 2021

Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): a population-based study.

Lancet 2020 08 11;396(10247):313-319. Epub 2020 Jun 11.

Division of Primary Care, Geneva University Hospitals, Geneva, Switzerland; Department of Health and Community Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Background: Assessing the burden of COVID-19 on the basis of medically attended case numbers is suboptimal given its reliance on testing strategy, changing case definitions, and disease presentation. Population-based serosurveys measuring anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies provide one method for estimating infection rates and monitoring the progression of the epidemic. Here, we estimate weekly seroprevalence of anti-SARS-CoV-2 antibodies in the population of Geneva, Switzerland, during the epidemic.

Methods: The SEROCoV-POP study is a population-based study of former participants of the Bus Santé study and their household members. We planned a series of 12 consecutive weekly serosurveys among randomly selected participants from a previous population-representative survey, and their household members aged 5 years and older. We tested each participant for anti-SARS-CoV-2-IgG antibodies using a commercially available ELISA. We estimated seroprevalence using a Bayesian logistic regression model taking into account test performance and adjusting for the age and sex of Geneva's population. Here we present results from the first 5 weeks of the study.

Findings: Between April 6 and May 9, 2020, we enrolled 2766 participants from 1339 households, with a demographic distribution similar to that of the canton of Geneva. In the first week, we estimated a seroprevalence of 4·8% (95% CI 2·4-8·0, n=341). The estimate increased to 8·5% (5·9-11·4, n=469) in the second week, to 10·9% (7·9-14·4, n=577) in the third week, 6·6% (4·3-9·4, n=604) in the fourth week, and 10·8% (8·2-13·9, n=775) in the fifth week. Individuals aged 5-9 years (relative risk [RR] 0·32 [95% CI 0·11-0·63]) and those older than 65 years (RR 0·50 [0·28-0·78]) had a significantly lower risk of being seropositive than those aged 20-49 years. After accounting for the time to seroconversion, we estimated that for every reported confirmed case, there were 11·6 infections in the community.

Interpretation: These results suggest that most of the population of Geneva remained uninfected during this wave of the pandemic, despite the high prevalence of COVID-19 in the region (5000 reported clinical cases over <2·5 months in the population of half a million people). Assuming that the presence of IgG antibodies is associated with immunity, these results highlight that the epidemic is far from coming to an end by means of fewer susceptible people in the population. Further, a significantly lower seroprevalence was observed for children aged 5-9 years and adults older than 65 years, compared with those aged 10-64 years. These results will inform countries considering the easing of restrictions aimed at curbing transmission.

Funding: Swiss Federal Office of Public Health, Swiss School of Public Health (Corona Immunitas research program), Fondation de Bienfaisance du Groupe Pictet, Fondation Ancrage, Fondation Privée des Hôpitaux Universitaires de Genève, and Center for Emerging Viral Diseases.
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http://dx.doi.org/10.1016/S0140-6736(20)31304-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289564PMC
August 2020

Special Report: The Biology of Inequalities in Health: The Lifepath Consortium.

Front Public Health 2020 12;8:118. Epub 2020 May 12.

Unit of Population Epidemiology, Division of Primary Care, Geneva University Hospitals, Geneva, Switzerland.

Funded by the European Commission Horizon 2020 programme, the research consortium aimed to investigate the effects of socioeconomic inequalities on the biology of healthy aging. The main research questions included the impact of inequalities on health, the role of behavioral and other risk factors, the underlying biological mechanisms, the efficacy of selected policies, and the general implications of our findings for theories and policies. The project adopted a life-course and comparative approach, considering lifetime effects from childhood and adulthood, and pooled data on up to 1.7 million participants of longitudinal cohort studies from Europe, USA, and Australia. These data showed that socioeconomic circumstances predicted mortality and functional decline as strongly as established risk factors currently targeted by global prevention programmes. Analyses also looked at socioeconomically patterned biological markers, allostatic load, and DNA methylation using richly phenotyped cohorts, unraveling their association with aging processes across the life-course. studies suggest that socioeconomic circumstances are embedded in our biology from the outset-i.e., disadvantage influences biological systems from molecules to organs. Our findings have important implications for policy, suggesting that (a) intervening on unfavorable socioeconomic conditions is complementary and as important as targeting well-known risk factors, such as tobacco and alcohol consumption, low fruit and vegetable intake, obesity and a sedentary lifestyle, and that (b) effects of preventive interventions in early life integrate interventions in adulthood. The report has an executive summary that refers to the different sections of the main paper.
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http://dx.doi.org/10.3389/fpubh.2020.00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235337PMC
May 2021

Ambulatory Blood Pressure in Relation to Plasma and Urinary Manganese.

Hypertension 2020 04 2;75(4):1133-1139. Epub 2020 Mar 2.

From the Department of Epidemiology and Health Systems, Center for Primary Care and Public Health-Unisanté (Z.-Y.Z., C.C., B.P., D.P., M. Bochud), University of Lausanne, Switzerland.

The association of blood pressure (BP) with manganese-an essential trace element required for human health-remains poorly studied. In 734 randomly recruited Swiss participants (mean age, 47.5 years; 51.4% women), we related ambulatory BP to 2 biomarkers, plasma manganese (pMn) and the urinary manganese (uMn) excretion. To allow for diurnal variation, we assessed BP and uMn over 24 hours and during wakefulness and sleep, using split urine samples. Twenty-four-hour, daytime, and nighttime systolic/diastolic BPs averaged 119.8/78.1, 123.8/81.2, and 107.0/68.3 mm Hg; the corresponding median uMn were 199.5, 83.0, and 51.5 μmol and median pMn, 0.52 μg/L. In analyses dichotomized by the median of the biomarkers, greater pMn was associated with higher 24-hour systolic/diastolic BP (+4.1/+2.3 mm Hg; ≤0.0003), greater daytime uMn with lower daytime BP (-3.5/-1.9 mm Hg; ≤0.0067), and greater nighttime uMn with higher nighttime BP (+2.9/+1.2 mm Hg; ≤0.046). In multivariable-adjusted analyses, significance (≤0.030) was retained for the positive association of 24-hour and daytime diastolic BP with pMn and for systolic BP in relation to uMn at night. The association sizes for a 2-fold increment in the biomarkers amounting to 0.77 mm Hg (95% CI, 0.08-1.47 mm Hg), 0.97 (CI, 0.20-1.76) and 1.33 (CI, 0.20-2.50 mm Hg), respectively. In conclusion, there were positive associations between diastolic BP and pMn over 24 hours and during daytime and between systolic BP and uMn at night.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.13649DOI Listing
April 2020

Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes.

Proc Natl Acad Sci U S A 2020 03 20;117(10):5310-5318. Epub 2020 Feb 20.

Department of Biomolecular Sciences, Weizmann Institute of Science, 7610001 Rehovot, Israel;

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αβα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αβα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.
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http://dx.doi.org/10.1073/pnas.1911742117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071883PMC
March 2020

Childhood socioeconomic conditions are associated with increased chronic low-grade inflammation over adolescence: findings from the EPITeen cohort study.

Arch Dis Child 2020 07 22;105(7):677-683. Epub 2020 Jan 22.

UNISANTE, Départment d'Épidémiologie et Systems de Santé, Lausanne, Switzerland.

Objective: Early life adversity has been associated with increased risk of inflammation and inflammation-related diseases in adulthood. This study aimed to examine the association of childhood socioeconomic conditions with chronic low-grade inflammation over adolescence.

Methods: We used information on 2942 members (1507 girls and 1435 boys) of the EPITeen (Epidemiological Health Investigation of Teenagers in Porto) cohort that was established in 2003 in Porto, Portugal, and included 13-year-old adolescents were further evaluated at 17 and 21 years. Mother' and father's education and occupation were used as indicators of childhood socioeconomic conditions. High-sensitivity C reactive protein (hs-CRP) was measured at three points in time (13, 17 and 21 years). hs-CRP levels were categorised in tertiles separately for each wave; chronic low-grade inflammation in adolescence was defined as having hs-CRP levels in the highest tertile in at least two waves and never in the lowest tertile.

Results: Prevalence of chronic low-grade inflammation during adolescence was significantly higher among participants with low parental socioeconomic position. Low parental socioeconomic position was associated with chronic low-grade inflammation in adolescence, after adjustment for sex, perinatal and physical environment factors, health-related behaviours and health status in adolescence OR=1.6; 95% CI: 1.1 to 2.4 for lowest versus highest mother's education and OR=1.6; 95% CI: 1.1 to 2.3 for lowest versus highest father's occupation.

Conclusion: Low childhood socioeconomic conditions are associated with chronic low-grade inflammation during adolescence. Our results suggest that the early life socioeconomic environment has an impact on inflammatory processes over adolescence.
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http://dx.doi.org/10.1136/archdischild-2019-317525DOI Listing
July 2020

Différences socio-économiques dans la Cohorte Lausannoise CoLaus.

Praxis (Bern 1994) 2020 Jan;109(1):23-26

Unisanté, Département d'épidémiologie et systèmes de santé, Lausanne.

Socio-Economic Differences in the Lausanne CoLaus Cohort The CoLaus study allowed to highlight the existence of broad social inequalities in health among the population of the city of Lausanne. In fact, participants with low socioeconomic status had a higher prevalence of cardio-metabolic risk factors, risk behaviors, sleep disturbances, and higher inflammatory markers compared to the more socio-economically advantaged participants in the study. In most cases, these inequalities are similar to those found in the neighboring cantons and countries.
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http://dx.doi.org/10.1024/1661-8157/a003364DOI Listing
January 2020

Mechanisms of life-course socioeconomic inequalities in adult systemic inflammation: Findings from two cohort studies.

Soc Sci Med 2020 01 19;245:112685. Epub 2019 Nov 19.

Center for Primary Care and Public Health (Unisanté), University of Lausanne, Switzerland; Unit of Population Epidemiology, Division of Primary Care, Geneva University Hospitals, Switzerland.

Disadvantaged socioeconomic conditions in childhood heighten systemic inflammatory levels in adulthood; however, life-course mechanisms underlying this association are largely unknown. In the present observational study, we investigated the roles of adulthood socioeconomic and lifestyle factors in mediating this association. Participants were from two prospective Swiss population-based cohorts (N = 5,152, mean age 60 years). We estimated the total effect of paternal occupational position on adult heightened systemic inflammatory levels (C-reactive protein>3 mg/L), and the indirect effects via adulthood socioeconomic positions (SEPs: education and occupational position), financial hardship, and lifestyle factors (body mass index, smoking status, physical inactivity, and alcohol consumption). We estimated odds ratio (OR) and proportion mediated using counterfactual-based mediation models. Individuals whose father had a low occupational position had an OR of 1.51 [95% confidence interval (CI): 1.25, 1.84] for heightened inflammation compared to their more advantaged counterparts. This was jointly mediated (33 [95% CI: 14, 69]%) by adulthood SEPs, whereby the pathway through education followed by occupational position mediated 30 [95% CI: 11, 64]%, while the pathway via occupational position only mediated 3 [95% CI: 4, 13]%. Individuals with the lowest life-course SEPs had an OR of 2.27 [95% CI: 1.71, 2.98] for heightened inflammation compared to having the highest life-course SEPs. This was jointly mediated (63 [95% CI: 44, 97]%) by financial hardship and lifestyle factors. Our study supports a cumulative effect of life-course SEPs on adult heightened systemic inflammation along the pathway paternal occupational position -> education -> adult occupational position. Financial hardship and lifestyle factors in adulthood mediate half of that effect.
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http://dx.doi.org/10.1016/j.socscimed.2019.112685DOI Listing
January 2020

The contribution of sleep to social inequalities in cardiovascular disorders: a multi-cohort study.

Cardiovasc Res 2020 07;116(8):1514-1524

Centre universitaire de médecine Générale et santé publique (UNISANTÉ), Institute of Social and Preventive Medicine (IUMSP), Route de la Corniche 10, 1010 Lausanne, Switzerland.

Aims: Sleep disturbances exhibit a strong social patterning, and inadequate sleep has been associated with adverse health outcomes, including cardiovascular disorders (CVD). However, the contribution of sleep to socioeconomic inequalities in CVD is unclear. This study pools data from eight European cohorts to investigate the role of sleep duration in the association between life-course socioeconomic status (SES) and CVD.

Methods And Results: We used cross-sectional data from eight European cohorts, totalling 111 205 participants. Life-course SES was assessed using father's and adult occupational position. Self-reported sleep duration was categorized into recommended (6-8.5 h/night), long (>8.5 h/night), and short (<6 h/night). We examined two cardiovascular outcomes: coronary heart disease (CHD) and stroke. Main analyses were conducted using pooled data and examined the association between life-course SES and CVD, and the contribution of sleep duration to this gradient using counterfactual mediation. Low father's occupational position was associated with an increased risk of CHD (men: OR = 1.19, 95% CI [1.04; 1.37]; women: OR = 1.25, 95% CI [1.02; 1.54]), with marginal decrease of the gradient after accounting for adult occupational position (men: OR = 1.17, 95% CI [1.02; 1.35]; women: OR = 1.22, 95% CI [0.99; 1.52]), and no mediating effect by short sleep duration. Low adult occupational position was associated with an increased risk of CHD in both men and women (men: OR = 1.48, 95% CI [1.14; 1.92]; women: OR = 1.53, 95% CI [1.04; 2.21]). Short sleep duration meaningfully contributed to the association between adult occupational position and CHD in men, with 13.4% mediation. Stroke did not exhibit a social patterning with any of the variables examined.

Conclusion: This study suggests that inadequate sleep accounts to a meaningful proportion of the association between adult occupational position and CHD, at least in men. With sleep increasingly being considered an important cardiovascular risk factor in its own terms, our study additionally points to its potential role in social inequalities in cardiovascular disease.
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http://dx.doi.org/10.1093/cvr/cvz267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425783PMC
July 2020

Interaction of carbohydrate-binding modules with poly(ethylene terephthalate).

Appl Microbiol Biotechnol 2019 Jun 16;103(12):4801-4812. Epub 2019 Apr 16.

Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, D-52425, Jülich, Germany.

Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity. In this study, we identified novel PET binding CBMs and characterized the CBM-PET interplay. We developed a semi-quantitative method to detect CBMs bound to PET films. Screening of eight CBMs from diverse families for PET binding revealed one CBM that possesses a high affinity towards PET. Molecular dynamics (MD) simulations of the CBM-PET interface revealed tryptophan residues forming an aromatic triad on the peptide surface. Their interaction with phenyl rings of PET is stabilized by additional hydrogen bonds formed between amino acids close to the aromatic triad. Furthermore, the ratio of hydrophobic to polar contacts at the interface was identified as an important feature determining the strength of PET binding of CBMs. The interaction of CBM tryptophan residues with PET was confirmed experimentally by tryptophan quenching measurements after addition of PET nanoparticles to CBM. Our findings are useful for engineering PET hydrolyzing enzymes and may also find applications in functionalization of PET.
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http://dx.doi.org/10.1007/s00253-019-09760-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536475PMC
June 2019

Structural Studies of Autophagy-Related Proteins.

Methods Mol Biol 2019 ;1880:17-56

Institute of Complex Systems ICS-6 (Structural Biochemistry), Forschungszentrum Jülich, Jülich, Germany.

Information about the structure and dynamics of proteins is crucial for understanding their physiological functions as well as for the development of strategies to modulate these activities. In this chapter we will describe the work packages required to determine the three-dimensional structures of proteins involved in autophagy by using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Further we will provide instructions how to perform a molecular dynamics (MD) simulation using GABARAP as example protein.
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http://dx.doi.org/10.1007/978-1-4939-8873-0_2DOI Listing
June 2019

Social inequalities in sleep-disordered breathing: Evidence from the CoLaus|HypnoLaus study.

J Sleep Res 2019 10 25;28(5):e12799. Epub 2018 Nov 25.

Institute of Social and Preventive Medicine (IUMSP), Lausanne University Hospital, Lausanne, Switzerland.

Sleep-disordered breathing is a common condition, related to a higher cardiometabolic and neurocognitive risk. The main risk factors for sleep-disordered breathing include obesity, craniofacial characteristics, male sex and age. However, some studies have suggested that adverse socioeconomic circumstances and lifestyle-related behaviours such as smoking and alcohol use, may also be risk factors for sleep-disordered breathing. Here, we investigate the associations between socioeconomic status and sleep-disordered breathing, as measured by sleep apnea-hypopnea and oxygen desaturation indexes. Furthermore, we assess whether these associations are explained by lifestyle-related factors (smoking, sedentary behaviour, alcohol use and body mass index [BMI]). We used data from the CoLaus|HypnoLaus study, a population-based study including 2162 participants from Lausanne (Switzerland). Socioeconomic status was measured through occupation and education. Sleep-disordered breathing was assessed through polysomnography and measured using the apnea-hypopnea index (AHI: number of apnea/hypopnea events/hr: ≥15/≥30 events), and the ≥3% oxygen desaturation index (ODI: number of oxygen desaturation events/hr: ≥15/≥30 events). Lower occupation and education were associated with higher AHI and ODI (occupation: AHI30, odds ratio (OR) = 1.88, 95% confidence interval (CI) [1.07; 3.31]; ODI30, OR = 2.29, 95% CI [1.19; 4.39]; education: AHI30, OR = 1.21, 95% CI [0.85; 1.72]; ODI30, OR = 1.26, 95% CI [0.83; 1.91]). BMI was associated with socioeconomic status and AHI/ODI, and contributed to the socioeconomic gradient in SDB, with mediation estimates ranging between 43% and 78%. In this Swiss population-based study, we found that low socioeconomic status is a risk factor for sleep-disordered breathing, and that these associations are partly explained by BMI. These findings provide a better understanding of the mechanisms underlying social differences in sleep-disordered breathing and may help implement policies for identifying high-risk profiles for this disorder.
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http://dx.doi.org/10.1111/jsr.12799DOI Listing
October 2019

Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case.

J Am Chem Soc 2018 11 8;140(46):15889-15903. Epub 2018 Nov 8.

Department of Chemistry - BMC , Uppsala University , BMC Box 576, 751 23 Uppsala , Sweden.

Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 "grips" the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ∼7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from Saccharomyces cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed-loop conformation can be sufficient to abolish most of the catalytic activity; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple "open and shut" case and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis.
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http://dx.doi.org/10.1021/jacs.8b09378DOI Listing
November 2018

The evolution of multiple active site configurations in a designed enzyme.

Nat Commun 2018 09 25;9(1):3900. Epub 2018 Sep 25.

Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.
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http://dx.doi.org/10.1038/s41467-018-06305-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156567PMC
September 2018

Molecular modeling of conformational dynamics and its role in enzyme evolution.

Curr Opin Struct Biol 2018 10 8;52:50-57. Epub 2018 Sep 8.

Science for Life Laboratory, Department of Chemistry - BMC, Uppsala University, BMC Box 576, S-751 23 Uppsala, Sweden. Electronic address:

With increasing computational power, biomolecular simulations have become an invaluable tool for understanding enzyme mechanisms and the origins of enzyme catalysis. More recently, computational studies have started to focus on understanding how enzyme activity itself evolves, both in terms of enhancing the native or new activities on existing enzyme scaffolds, or completely de novo on previously non-catalytic scaffolds. In this context, both experiment and molecular modeling provided strong evidence for an important role of conformational dynamics in the evolution of enzyme functions. This contribution will present a brief overview of the current state of the art for computationally exploring enzyme conformational dynamics in enzyme evolution, and, using several showcase studies, illustrate the ways molecular modeling can be used to shed light on how enzyme function evolves, at the most fundamental molecular level.
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http://dx.doi.org/10.1016/j.sbi.2018.08.004DOI Listing
October 2018

Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis.

ACS Catal 2018 May 27;8(5):3971-3984. Epub 2018 Mar 27.

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of DERA (DERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the DERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, DERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the DERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis.
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http://dx.doi.org/10.1021/acscatal.7b04408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080863PMC
May 2018

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase.

ACS Omega 2018 Apr 2;3(4):3665-3674. Epub 2018 Apr 2.

Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, S-751 24 Uppsala, Sweden.

Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrate-Schiff base to the product-Schiff base, which includes the rate-limiting cleavage of the Cα-H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the Cα-H bond involves direct hydride transfer to the topaquinone cofactor-a mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu cation to the reaction site, lower the activation barrier to drive the chemical reaction.
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http://dx.doi.org/10.1021/acsomega.8b00346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044848PMC
April 2018

Conformational dynamics and enzyme evolution.

J R Soc Interface 2018 07;15(144)

Departamento de Quimica Fisica, Facultad de Ciencias, University of Granada, 18071 Granada, Spain

Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biological function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-of-the-art computational and experimental approaches are revealing the crucial molecular details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temperatures and the possibility of additional phenotypic mutations.
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http://dx.doi.org/10.1098/rsif.2018.0330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073641PMC
July 2018

How accurately do force fields represent protein side chain ensembles?

Proteins 2018 09;86(9):935-944

Institute of Complex Systems, Structural Biochemistry, Forschungszentrum Jülich, Jülich, 52425, Germany.

Although the protein backbone is the most fundamental part of the structure, the fine-tuning of side-chain conformations is important for protein function, for example, in protein-protein and protein-ligand interactions, and also in enzyme catalysis. While several benchmarks testing the performance of protein force fields for side chain properties have already been published, they often considered only a few force fields and were not tested against the same experimental observables; hence, they are not directly comparable. In this work, we explore the ability of twelve force fields, which are different flavors of AMBER, CHARMM, OPLS, or GROMOS, to reproduce average rotamer angles and rotamer populations obtained from extensive NMR studies of the J and residual dipolar coupling constants for two small proteins: ubiquitin and GB3. Based on a total of 196 μs sampling time, our results reveal that all force fields identify the correct side chain angles, while the AMBER and CHARMM force fields clearly outperform the OPLS and GROMOS force fields in estimating rotamer populations. The three best force fields for representing the protein side chain dynamics are AMBER 14SB, AMBER 99SB*-ILDN, and CHARMM36. Furthermore, we observe that the side chain ensembles of buried amino acid residues are generally more accurately represented than those of the surface exposed residues.
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http://dx.doi.org/10.1002/prot.25525DOI Listing
September 2018

The contribution of health behaviors to socioeconomic inequalities in health: A systematic review.

Prev Med 2018 08 9;113:15-31. Epub 2018 May 9.

Institute of Social and Preventive Medicine (IUMSP), Lausanne University Hospital, Route de la corniche 10, 1010 Lausanne, Switzerland.

Unhealthy behaviors and their social patterning have been frequently proposed as factors mediating socioeconomic differences in health. However, a clear quantification of the contribution of health behaviors to the socioeconomic gradient in health is lacking. This study systematically reviews the role of health behaviors in explaining socioeconomic inequalities in health. Published studies were identified by a systematic review of PubMed, Embase and Web-of-Science. Four health behaviors were considered: smoking, alcohol consumption, physical activity and diet. We restricted health outcomes to cardiometabolic disorders and mortality. To allow comparison between studies, the contribution of health behaviors, or the part of the socioeconomic gradient in health that is explained by health behaviors, was recalculated in all studies according to the absolute scale difference method. We identified 114 articles on socioeconomic position, health behaviors and cardiometabolic disorders or mortality from electronic databases and articles reference lists. Lower socioeconomic position was associated with an increased risk of all-cause mortality and cardiometabolic disorders, this gradient was explained by health behaviors to varying degrees (minimum contribution -43%; maximum contribution 261%). Health behaviors explained a larger proportion of the SEP-health gradient in studies conducted in North America and Northern Europe, in studies examining all-cause mortality and cardiovascular disease, among men, in younger individuals, and in longitudinal studies, when compared to other settings. Of the four behaviors examined, smoking contributed the most to social inequalities in health, with a median contribution of 19%. Health behaviors contribute to the socioeconomic gradient in cardiometabolic disease and mortality, but this contribution varies according to population and study characteristics. Nevertheless, our results should encourage the implementation of interventions targeting health behaviors, as they may reduce socioeconomic inequalities in health and increase population health.
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http://dx.doi.org/10.1016/j.ypmed.2018.05.003DOI Listing
August 2018

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States.

Biophys J 2018 04;114(7):1614-1623

Institute of Complex Systems, Structural Biochemistry, Research Center Jülich, Jülich, Germany; Institute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany. Electronic address:

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson's disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called "random coil conformation" consists of α-helical and β-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.
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http://dx.doi.org/10.1016/j.bpj.2018.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954275PMC
April 2018

Simulation-Guided Design of Cytochrome P450 for Chemo- and Regioselective Macrocyclic Oxidation.

J Chem Inf Model 2018 04 23;58(4):848-858. Epub 2018 Mar 23.

Institute of Complex Systems: Structural Biochemistry , Forschungszentrum Jülich , 52425 Jülich , Germany.

Engineering high chemo-, regio-, and stereoselectivity is a prerequisite for enzyme usage in organic synthesis. Cytochromes P450 can oxidize a broad range of substrates, including macrocycles, which are becoming popular scaffolds for therapeutic agents. However, a large conformational space explored by macrocycles not only reduces the selectivity of oxidation but also impairs computational enzyme design strategies based on docking and molecular dynamics (MD) simulations. We present a novel design workflow that uses enhanced-sampling Hamiltonian replica exchange (HREX) MD and focuses on quantifying the substrate binding for suggesting the mutations to be made. This computational approach is applied to P450 BM3 with the aim to shift regioselectively toward one of the numerous possible positions during β-cembrenediol oxidation. The predictions are experimentally tested and the resulting product distributions validate our design strategy, as single mutations led up to 5-fold regioselectivity increases. We thus conclude that the HREX-MD-based workflow is a promising tool for the identification of positions for mutagenesis aiming at P450 enzymes with improved regioselectivity.
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http://dx.doi.org/10.1021/acs.jcim.8b00043DOI Listing
April 2018

Challenges and advances in the computational modeling of biological phosphate hydrolysis.

Chem Commun (Camb) 2018 Mar 7;54(25):3077-3089. Epub 2018 Feb 7.

Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, S-751 24 Uppsala, Sweden.

Phosphate ester hydrolysis is fundamental to many life processes, and has been the topic of substantial experimental and computational research effort. However, even the simplest of phosphate esters can be hydrolyzed through multiple possible pathways that can be difficult to distinguish between, either experimentally, or computationally. Therefore, the mechanisms of both the enzymatic and non-enzymatic reactions have been historically controversial. In the present contribution, we highlight a number of technical issues involved in reliably modeling these computationally challenging reactions, as well as proposing potential solutions. We also showcase examples of our own work in this area, discussing both the non-enzymatic reaction in aqueous solution, as well as insights obtained from the computational modeling of organophosphate hydrolysis and catalytic promiscuity amongst enzymes that catalyze phosphoryl transfer.
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http://dx.doi.org/10.1039/c7cc09504jDOI Listing
March 2018

Shuffling Active Site Substate Populations Affects Catalytic Activity: The Case of Glucose Oxidase.

ACS Catal 2017 Sep 1;7(9):6188-6197. Epub 2017 Aug 1.

Institute of Complex Systems: Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany.

Glucose oxidase has wide applications in the pharmaceutical, chemical, and food industries. Many recent studies have enhanced key properties of this enzyme using directed evolution, yet without being able to reveal why these mutations are actually beneficial. This work presents a synergistic combination of experimental and computational methods, indicating how mutations, even when distant from the active site, positively affect glucose oxidase catalysis. We have determined the crystal structures of glucose oxidase mutants containing molecular oxygen in the active site. The catalytically important His516 residue has been previously shown to be flexible in the wild-type enzyme. The molecular dynamics simulations performed in this work allow us to quantify this floppiness, revealing that His516 exists in two states: catalytic and noncatalytic. The relative populations of these two substates are almost identical in the wild-type enzyme, with His516 readily shuffling between them. In the glucose oxidase mutants, on the other hand, the mutations enrich the catalytic His516 conformation and reduce the flexibility of this residue, leading to an enhancement in their catalytic efficiency. This study stresses the benefit of active site preorganization with respect to enzyme conversion rates by reducing molecular reorientation needs. We further suggest that the computational approach based on Hamiltonian replica exchange molecular dynamics, used in this study, may be a general approach to screening in silico for improved enzyme variants involving flexible catalytic residues.
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http://dx.doi.org/10.1021/acscatal.7b01575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745072PMC
September 2017
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