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Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide and is often associated with aspects of metabolic syndrome. Despite its prevalence and the importance of early diagnosis, there is a lack of robustly validated biomarkers for diagnosis, prognosis and monitoring of disease progression in response to a given treatment. In this Review, we provide an overview of the contribution of metabolomics and lipidomics in clinical studies to identify biomarkers associated with NAFLD and nonalcoholic steatohepatitis (NASH). In addition, we highlight the key metabolic pathways in NAFLD and NASH that have been identified by metabolomics and lipidomics approaches and could potentially be used as biomarkers for non-invasive diagnostic tests. Overall, the studies demonstrated alterations in amino acid metabolism and several aspects of lipid metabolism including circulating fatty acids, triglycerides, phospholipids and bile acids. Although we report several studies that identified potential biomarkers, few have been validated. Metabolomics and lipidomics approaches are being used to identify biomarkers for nonalcoholic fatty liver disease (NAFLD). This Review discusses the application of metabolomics and lipidomics in clinical studies and in the identification of key metabolic pathway alterations in NAFLD. Key points Nonalcoholic fatty liver disease (NAFLD) affects 25% of the adult world population; in about 20% of patients, it can progress to nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis. There is an urgent need for development of clinically relevant biomarkers and non-invasive diagnostic tests for NAFLD. Metabolomics and lipidomics approaches have provided insightful evidence of altered metabolic pathways in NAFLD and NASH. There is an association between circulating amino acids and steatohepatitis, and impairment in amino acid metabolism in NAFLD is strongly correlated with insulin resistance, particularly in the muscle. An increase in oxidative stress results in a reduction in hepatic glutathione levels and is associated with liver damage and the progression of NAFLD to NASH. NASH is strongly associated with alterations in circulating fatty acids and intact lipids, which is partially due to alterations in de novo liver lipogenesis, lipolysis rate and VLDL metabolism.

Understanding the distribution and variation in inflammatory markers is crucial for advancing our knowledge of inflammatory processes and evaluating their clinical utility in diagnosing and monitoring acute and chronic disease. 1 H NMR spectroscopy of blood plasma and serum was applied to measure a composite panel of inflammatory markers based on acute phase glycoprotein signals (GlycA and GlycB) and sub-regions of the lipoprotein derived Supramolecular Phospholipid Composite signals (SPC 1 , SPC 2 and SPC 3 ) to establish normal ranges in two healthy, predominantly white cohorts from Australia (n = 398) and Spain (n = 80; ages 20–70 years). GlycA, GlycB, SPC 1 and SPC 3 were not significantly impacted by age or sex, but SPC 2 (an HDL-related biomarker) was significantly higher in women across all age ranges by an average of 33.7%. A free-living Australian population cohort (n = 3945) was used to explore the relationship of BMI with the panel of inflammatory markers. The glycoprotein signals were directly associated with BMI with GlycB levels being significantly higher for women in all BMI classes. Conversely, SPC 2 was found to be inversely associated with BMI and differed significantly between the sexes at each BMI category (normal weight p = 3.46x10 -43 , overweight p = 3.33x10 -79 , obese p = 2.15x10 -64 ). SPC 1 and SPC 3 were markedly less affected by BMI changes. Given the significant association between SPC 2 and sex, these data suggest that men and women should be modelled independently for NMR-determined inflammatory biomarkers, or that data should be corrected for sex.

Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA. Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups.

Adeno-associated viruses (AAVs) are promising gene therapy vectors, but challenges arise when treating patients with preexisting neutralizing antibodies. Worldwide seroprevalence studies provide snapshots of existing immunity in diverse populations. Owing to the uniqueness of the Basque socio-geographical landscape, we investigated the seroprevalence of eight AAV serotypes in residents of the Basque Country. We found the highest seroprevalence of AAV3, and the lowest seroprevalence of AAV9. Additionally, less than 50% of the Basque population has neutralizing antibodies against AAV4, AAV6, and AAV9. Our findings provide insight into AAV infections in the Basque region, public health, and the development of AAV-based therapeutics.

Background Reliable non-invasive biomarkers for endometriosis remain unavailable in routine practice, and their translational value depends on performance in symptomatic referral populations rather than only against healthy controls. We evaluated Nuclear Magnetic Resonance (NMR)-based serum metabolite and lipoprotein profiling for endometriosis across clinically relevant and physiological comparator settings, alongside exploratory analyses of systemic biological variation. Methods Blood serum samples from women with surgically confirmed endometriosis, symptomatic controls, and healthy volunteers underwent quantitative in vitro diagnostics research (IVDr) 1 H-NMR-based metabolite and lipoprotein profiling. A subset also underwent cytokine profiling. Two diagnostic settings were prespecified: endometriosis versus symptomatic controls (primary) and endometriosis versus healthy volunteers (secondary). Baseline models included age and body mass index, while full models incorporated the IVDr metabolite-lipoprotein panel using elastic net regularization. Performance was assessed using fully nested repeated cross-validation and an independently processed temporal cohort. Exploratory analyses included covariate-adjusted group comparisons, weighted correlation network analysis, cytokine correlations, and paired pre-/post-operative comparisons. Results In the primary symptomatic-control comparison, the IVDr panel did not improve diagnostic performance beyond age and body mass index (AUC 0.620 vs. 0.637 for baseline). Discrimination was substantially higher in the healthy-volunteer comparison (AUC 0.994 for the full model vs. 0.882 for baseline), but this pattern was not reproduced in the temporal cohort, where performance was poor in both comparator settings. Exploratory analyses showed that the clearest biological differences were concentrated in healthy-based contrasts, with lower amino acids, creatinine, lactic acid, and selected low-density lipoprotein (LDL) measures in endometriosis. Part of the amino-acid pattern was also present in symptomatic controls, whereas particularly LDL6 lipoprotein subfractions, appeared more restricted and were supported by lipoprotein-enriched network structure. Cytokine-cytokine correlations showed reproducible within-panel immune covariance, but no cross-domain correlations remained significant after false discovery rate correction. Conclusions While NMR-based serum metabolite and lipoprotein profiling showed strong apparent discrimination against healthy volunteers, performance was limited in the clinically relevant symptomatic-control setting, underscoring the importance of comparator spectrum for translational biomarker evaluation. Exploratory analyses identified biologically informative serum patterns, particularly a more restricted lipoprotein-subclass LDL6 signal that warrants targeted replication in clinically representative and analytically harmonized studies.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid β-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease. Lower activity of MATα1, which catalyzes the synthesis of the methyl donor S-adenosylmethionine, and mitochondrial dysfunction occur in alcohol-associated liver disease (ALD). Here the authors report that the peptidyl-prolyl cis/trans isomerase PIN1 mediates a selective depletion of MATα1 in the mitochondria, which contributes to mitochondrial dysfunction and fat accumulation, in mouse models of ALD.

Hypertension is a highly prevalent medical condition that occurs when blood pressure is too high, which greatly increases the risk of developing other cardiovascular diseases and is generally associated with higher rates of morbidity and mortality. Due to the silent/asymptomatic nature of hypertension, although the methods currently available to diagnose it are easy, they generally do not allow for an early diagnosis and an efficient prognosis to avoid irreversible damage in the medium or long term. In fact, an early diagnosis of hypertension would be crucial to decrease hypertension-associated mortality. Since metabolomics using NMR can provide a global measurement of various serum metabolites, it is very suitable for detecting novel biomarkers. We therefore analyzed serum metabolomic profiles among normotensive and hypertensive elderly individuals by NMR and identified new potential biomarkers for hypertension and associated diseases. We found higher levels of acetate, formate, and glycerol, and lower levels of glutamine, glycine, and sarcosine in individuals with hypertension. Therefore, these metabolites could be used for early diagnosis of hypertension to avoid comorbidities derived from hypertension and associated mortality.

Split personality Here's a new way to look at familiar enzymes. A new technology that can detect ultra-rare states of a protein (cyclophilin A in this instance) shows that rather than having a range of conformations to which it resorts during catalysis, it has them all before it starts. Protein motions needed for catalysis are intrinsic to the enzyme and take in the whole molecule, not just the traditional centre of attention, the active site. A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis 1 , 2 , 3 , 4 , 5 . The concept of conformational substates has been described 6 ; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis – trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously 3 , 7 , 8 , 9 , 10 , we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design. Targeting biomedically relevant protein-protein interactions is a long-lasting challenge in medicinal chemistry. Here, the authors develop a single α-helical peptide scaffold that can be tailored to target globular proteins of biomedical interest.