Microsecond Molecular Dynamics Simulations of Intrinsically Disordered Proteins Involved in the Oxidative Stress Response

Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5–1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response

Circulating triacylglycerol signatures and insulin sensitivity in NAFLD associated with the E167K variant in TM6SF2

Background & Aims The Glu167Lys (E167K) variant in the transmembrane 6 superfamily member 2 protein (TM6SF2) was recently shown to influence liver fat (LFAT) content. We aimed at studying how this variant influences circulating triacylglycerol (TAG) signatures and whether it influences hepatic or adipose tissue insulin sensitivity. Methods We genotyped 300 Finnish subjects for the E167K (rs58542926) variant in TM6SF2 and for the I148M (rs738409) variant in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) in whom LFAT was measured using 1H-MRS and circulating lipids by UPLC-MS. We compared the plasma lipidome between E167K carriers (TM6SF2EK/KK) and non-carriers (TM6SF2EE), and between three groups of NAFLD: (i) carriers of the E167K but not of the I148M variant in PNPLA3 (‘TM6SF2 NAFLD’), (ii) carriers of the I148M but not of the E167K variant (‘PNPLA3 NAFLD’), and (iii) non-carriers of either risk allele (‘Non-risk NAFLD’). Hepatic and adipose tissue insulin sensitivities were measured using the euglycemic hyperinsulinemic clamp technique combined with infusion of [3-3H]glucose in 111 subjects. Results The LFAT content was 34% higher in the TM6SF2EK/KK (13.07 ± 1.57%) than in the TM6SF2EE group (9.77 ± 0.58%, p = 0.013). The effect of insulin on glucose production and lipolysis were significantly higher in the TM6SF2EK/KK than in the TM6SF2EE group. Comparison of the three NAFLD groups with similar LFATs showed that both the ‘TM6SF2 NAFLD’ and ‘PNPLA3 NAFLD’ had significantly lower triglyceride levels and were characterized by lower levels of most common TAGs compared to the ‘Non-risk NAFLD’ group. Conclusions We conclude that the E167K variant in TM6SF2 is associated with a distinct subtype of NAFLD, characterized by preserved insulin sensitivity with regard to lipolysis, hepatic glucose production and lack of hypertriglyceridemia despite a clearly increased LFAT content

Lipidomics in biomedical research-practical considerations

Lipids have many central physiological roles including as structural components of cell membranes, energy storage sources and intermediates in signaling pathways. Lipid-related disturbances are known to underlie many diseases and their co-morbidities. The emergence of lipidomics has empowered researchers to study lipid metabolism at the cellular as well as physiological levels at a greater depth than was previously possible. The key challenges ahead in the field of lipidomics in medical research lie in the development of experimental protocols and in silico techniques needed to study lipidomes at the systems level. Clinical questions where lipidomics may have an impact in healthcare settings also need to be identified, both from the health outcomes and health economics perspectives. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.Peer reviewe

Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis.

BACKGROUND: Lipids are an important and highly diverse class of molecules having structural, energy storage and signaling roles. Modern analytical technologies afford screening of many lipid molecular species in parallel. One of the biggest challenges of lipidomics is elucidation of important pathobiological phenomena from the integration of the large amounts of new data becoming available. RESULTS: We present computational and informatics approaches to study lipid molecular profiles in the context of known metabolic pathways and established pathophysiological responses, utilizing information obtained from modern analytical technologies. In order to facilitate identification of lipids, we compute the scaffold of theoretically possible lipids based on known lipid building blocks such as polar head groups and fatty acids. Each compound entry is linked to the available information on lipid pathways and contains the information that can be utilized for its automated identification from high-throughput UPLC/MS-based lipidomics experiments. The utility of our approach is demonstrated by its application to the lipidomic characterization of the fatty liver of the genetically obese insulin resistant ob/ob mouse model. We investigate the changes of correlation structure of the lipidome using multivariate analysis, as well as reconstruct the pathways for specific molecular species of interest using available lipidomic and gene expression data. CONCLUSION: The methodology presented herein facilitates identification and interpretation of high-throughput lipidomics data. In the context of the ob/ob mouse liver profiling, we have identified the parallel associations between the elevated triacylglycerol levels and the ceramides, as well as the putative activated ceramide-synthesis pathways.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Decreased cord-blood phospholipids in young age at onset type 1 diabetes.

Children developing type 1 diabetes may have risk markers already in their umbilical cord blood. It is hypothesized that the risk for type 1 diabetes at an early age may be increased by a pathogenic pregnancy and be reflected in altered cord-blood composition. In this study metabolomics was used to test if the cord-blood lipidome was affected in children diagnosed with type 1 diabetes before eight years of age. The present case-control study of 76 index children diagnosed with type 1 diabetes before eight years of age and 76 healthy controls matched for HLA risk, gender and date of birth as well as mother's age and gestational age revealed that cord-blood phosphatidylcholines and phosphatidylethanolamines were significantly decreased in children diagnosed with type 1 diabetes before four years of age. Reduced levels of triglycerides correlated to gestational age in both index and control children and to age at diagnosis only in the index children. Finally, gestational infection during the first trimester was associated with lower cord blood total lysophosphatidylcholines in both index and control children. In conclusion, metabolomics of umbilical cord blood may identify children at increased risk for type 1 diabetes. Low phospholipid levels at birth may represent key mediators of the immune system and contribute in early induction of islet autoimmunity

The Gut Microbiota Modulates Glycaemic Control and Serum Metabolite Profiles in Non-Obese Diabetic Mice

Peer reviewe

Hepatic ceramides dissociate steatosis and insulin resistance in patients with non-alcoholic fatty liver disease

Background & Aims: Recent data in mice have identified de novo ceramide synthesis as the key mediator of hepatic insulin resistance (IR) that in humans characterizes increases in liver fat due to IR ('Metabolic NAFLD' but not that due to the I148M gene variant in PNPLA3 ('PNPLA3 NAFLD'). We determined which bioactive lipids co-segregate with IR in the human liver. Methods: Liver lipidome was profiled in liver biopsies from 125 subjects that were divided into equally sized groups based on median HOMA-IR ('High and Low HOMA-IR', n = 62 and n = 63) or PNPLA3 genotype (PNPIA3(148MM/MI), n = 61 vs. PNPLA3(148II), n = 64). The subjects were also divided into 4 groups who had either IR, the I148M gene variant, both of the risk factors or neither. Results: Steatosis and NASH prevalence were similarly increased in 'High HOMA-IR' and PNPLA3(148MM/MI) groups compared to their respective control groups. The 'High HOMA-IR' but not the PNPLA3(148MM/MI) group had features of IR. The liver in 'High HOMA-IR' vs. low HOMA-IR' was markedly enriched in saturated and monounsaturated triacylglycerols and free fatty acids, dihydroceramides (markers of de novo ceramide synthesis) and ceramides. Markers of other ceramide synthetic pathways were unchanged. In PNPLA3(148MM/MI) vs. PNPLA3(148II), the increase in liver fat was due to polyunsaturated triacylglycerols while other lipids were unchanged. Similar changes were observed when data were analyzed using the 4 subgroups. Conclusions: Similar increases in liver fat and NASH are associated with a metabolically harmful saturated, ceramide-enriched liver lipidome in 'Metabolic NAFLD' but not in 'PNPLA3 NAFLD'. This difference may explain why metabolic but not PNPLA3 NAFLD increases the risk of type 2 diabetes and cardiovascular disease. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.Peer reviewe

Lipidome as a predictive tool in progression to type 2 diabetes in Finnish men

Background. There is a need for early markers to track and predict the development of type 2 diabetes mellitus (T2DM) from the state of normal glucose tolerance through prediabetes. In this study we tested whether the plasma molecular lipidome has biomarker potential to predicting the onset of T2DM. Methods. We applied global lipidomic profiling on plasma samples from well-phenotyped men (107 cases, 216 controls) participating in the longitudinal METSIM study at baseline and at five-year follow-up. To validate the lipid markers, an additional study with a representative sample of adult male population (n = 631) was also conducted. A total of 277 plasma lipids were analyzed using the lipidomics platform based on ultra performance liquid chromatography coupled to time-of-flight mass spectrometry. Lipids with the highest predictive power for the development of T2DM were computationally selected, validated and compared to standard risk models without lipids. Results. A persistent lipid signature with higher levels of triacylglycerols and diacyl-phospholipids as well as lowerlevels of alkylacyl phosphatidylcholines was observed in progressors to T2DM. Lysophosphatidylcholine acyl C18:2 (LysoPC(18:2)), phosphatidylcholines PC(32:1), PC(34:2e) and PC(36:1), and triacylglycerol TG(17:1/18:1/18:2) were selected to the full model that included metabolic risk factors and FINDRISC variables. When further adjusting for BM and age, these lipids had respective odds ratios of 0.32, 2.4, 0.50, 2.2 and 0.31 (all p 0; p <0.05). Notably, the lipid models remained predictive of the development of T2DM in the fasting plasma glucose-matched subset of the validation study. Conclusion. This study indicates that a lipid signature characteristic of T2DM is present years before the diagnosis and improves prediction of progression to T2DM. Molecular lipid biomarkers were shown to have predictive power also in a high-risk group, where standard risk factors are not helpful at distinguishing progressors from non-progressors. (C) 2017 The Authors. Published by Elsevier Inc.Peer reviewe