The presentation of peptides on class I human leukocyte antigen (HLA-I) molecules plays a central role in immune recognition of infected or malignant cells. In cancer, non-self HLA-I ligands can arise from many different alterations, including non-synonymous mutations, gene fusion, cancer-specific alternative mRNA splicing or aberrant post-translational modifications. Identifying HLA-I ligands remains a challenging task that requires either heavy experimental work for in vivo identification or optimized bioinformatics tools for accurate predictions. To date, no HLA-I ligand predictor includes post-translational modifications. To fill this gap, we curated phosphorylated HLA-I ligands from several immunopeptidomics studies (including six newly measured samples) covering 72 HLA-I alleles and retrieved a total of 2,066 unique phosphorylated peptides. We then expanded our motif deconvolution tool to identify precise binding motifs of phosphorylated HLA-I ligands. Our results reveal a clear enrichment of phosphorylated peptides among HLA-C ligands and demonstrate a prevalent role of both HLA-I motifs and kinase motifs on the presentation of phosphorylated peptides. These data further enabled us to develop and validate the first predictor of interactions between HLA-I molecules and phosphorylated peptides.

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.

The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. To support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.

The immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available.

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Two new regions of Microsoft's Azure cloud will open in Canberra on Tuesday.

Corporate Members Event Nominees Breakfast Briefing Partners and Major Corporates

Event participants

Christopher Sabatini, Senior Research Fellow for Latin America, US and the Americas Programme, Chatham House

3 October 2019

Research Event

Event participants

Michael Gasiorek, Professor of Economics, University of Sussex; Director, Interanalysis; Fellow, UK Trade Policy Observatory, University of Sussex
Julia Magntorn Garrett, Research Officer, UK Trade Policy Observatory, University of Sussex
Prof Jim Rollo, Deputy Director, UK Trade Policy Observatory, University of Sussex; Associate Fellow, Global Economy and Finance Department, Chatham House
Nicolo Tamberi, Research Officer in the Economics of Brexit, University of Sussex
L. Alan Winters, Professor of Economics, Director, UK Trade Policy Observatory, University of Sussex

Members Event Webinar

Event participants

Pepijn Bergsen, Research Fellow, Europe Programme, Chatham House

Dr Sheri Berman, Professor of Political Science, Barnard College

Chair: Hans Kundnani, Senior Research Fellow, Europe Programme, Chatham House

Join host Scott Carey as the team dissects Tory meltdown and what it means for tech and the ministers we haven't heard of. What can they actually do to help the country? Charlotte Jee explains.

Then Henry Burrell chats on the new Microsoft Surface Go, an 'affordable' Surface tablet that actually still breaks the bank. Who is it for, and is Microsoft really chasing the iPad market?

David Price rounds up the pod with Musk Corner as everyone's favourite Twitter megalomaniac flies off to Thailand to help with a cave rescue - but should he stay out of it?

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This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Atherogenic LDL particles are physicochemically and metabolically heterogeneous. Can bioactive lipid cargo differentiate LDL subclasses, and thus potential atherogenicity?  What is the effect of statin treatment? Obese, hypertriglyceridemic, hypercholesterolemic males (n=12; Lp(a) <10 mg/dL) received pitavastatin calcium (4mg/day) for 180 days in a single-phase, unblinded study. The lipidomic profiles (23 lipid classes) of five LDL subclasses fractionated from baseline and post-statin plasmas were determined by LC-MS. At baseline and on statin treatment, very small dense LDL (LDL5) was preferentially enriched (up to 3-fold) in specific lysophospholipids (lysophosphatidylcholine (LPC); lysophosphatidylinositol (LPI); lyso-platelet activating factor (LPC(O)); 9,0.2 and 0.14 mol/mol apoB respectively; all p<0.001 versus LDL1-4), suggesting  elevated inflammatory potential per particle. In contrast, lysophosphatidylethanolamine was uniformly distributed among LDL subclasses. Statin treatment markedly reduced absolute plasma concentrations of all LDL subclasses (up to 33.5%), including LPC, LPI and LPC(O) contents (up to -52%), consistent with reduction in cardiovascular risk. Despite such reductions, lipotoxic ceramide load per particle in LDL1-5 (1.5 - 3 mol/mol apoB; 3 - 7 mmol/mol phosphatidylcholine) was either conserved or elevated. Bioactive lipids may constitute biomarkers for the cardiometabolic risk associated with specific LDL subclasses in atherogenic dyslipidemia at baseline, and with residual risk on statin therapy.

31 March 2020

23 April 2020

Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed bio-molecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focusses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed which are related to this mechanism. Typical spectra of peptides, proteins and non-covalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent non-covalent complexes are described.

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range.  Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Whilst immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

1 March 2007 , Number 11

In early November, the retiring head of Britain’s Security Service MI5, Dame Eliza Manningham-Buller, warned that the danger of a terror attack was ‘serious’ and ‘growing’, with as many as thirty plots underway. Traditional terrorism of the sort practised by the Irish Republican Army has given way to the possibility, if not the expectation, that groups such as Al Qaeda might make use of chemical, biological, radiological and nuclear weapons and materials in an attack in Britain. So what are the dangers?

7 May 2020 , Volume 96, Number 3

Invitation Only Research Event

Event participants

Oscar Jonsson, Director, Stockholm Free World Forum (Frivärld)
Mathieu Boulègue, Research Fellow, Russia and Eurasia Programme, Chatham House
Chair: ​Alice Billon-Galland, Research Associate, Europe Programme, Chatham House

27 November 2019

Invitation Only Research Event

Event participants

Archbishop Yevstraty (Zoria) of Chernihiv, Deputy Head of Department for External Church Relations, Ukrainian Orthodox Church (Orthodox Church of Ukraine)

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Whether HDL is associated with dementia risk is unclear. In addition to apoA1, other apolipoproteins are found in HDL, creating subspecies of HDL that may have distinct metabolic properties. We measured apoA1, apoC3, and apoJ levels in plasma and apoA1 levels in HDL that contains or lacks apoE, apoJ, or apoC3 using a modified sandwich ELISA in a case-cohort study nested within the Ginkgo Evaluation of Memory Study. We included 995 randomly selected participants and 521 participants who developed dementia during a mean of 5.1 years of follow-up. The level of total apoA1 was not significantly related to dementia risk, regardless of the coexistence of apoC3, apoJ, or apoE. Higher levels of total plasma apoC3 were associated with better cognitive function at baseline (difference in Modified Mini-Mental State Examination scores tertile 3 vs. tertile 1: 0.60; 95% CI: 0.23, 0.98) and a lower dementia risk (adjusted hazard ratio tertile 3 vs. tertile 1: 0.73; 95% CI: 0.55, 0.96). Plasma concentrations of apoA1 in HDL and its apolipoprotein-defined subspecies were not associated with cognitive function at baseline or with the risk of dementia during follow-up. Similar studies in other populations are required to better understand the association between apoC3 and Alzheimer’s disease pathology.

Plasma lipoprotein (a) [Lp(a)] levels are largely determined by variation in the LPA gene, which codes for apo(a). Genome-wide association studies (GWASs) have identified nonsynonymous variants in LPA that associate with low Lp(a) levels, although their effect on apo(a) function is unknown. We investigated two such variants, R990Q and R1771C, which were present in four null Lp(a) individuals, for structural and functional effects. Sequence alignments showed the R990 and R1771 residues to be highly conserved and homologous to each other and to residues associated with plasminogen deficiency. Structural modeling showed both residues to make several polar contacts with neighboring residues that would be ablated on substitution. Recombinant expression of the WT and R1771C apo(a) in liver and kidney cells showed an abundance of an immature form for both apo(a) proteins. A mature form of apo(a) was only seen with the WT protein. Imaging of the recombinant apo(a) proteins in conjunction with markers of the secretory pathway indicated a poor transit of R1771C into the Golgi. Furthermore, the R1771C mutant displayed a glycosylation pattern consistent with ER, but not Golgi, glycosylation. We conclude that R1771 and the equivalent R990 residue facilitate correct folding of the apo(a) kringle structure and mutations at these positions prevent the proper folding required for full maturation and secretion. To our knowledge, this is the first example of nonsynonymous variants in LPA being causative of a null Lp(a) phenotype.

CVD is the leading cause of death worldwide, and genetic investigations into the human lipidome may provide insight into CVD risk. The aim of this study was to estimate the heritability of circulating lipid species and their genetic correlation with CVD traits. Targeted lipidomic profiling was performed on 4,492 participants from the Busselton Family Heart Study to quantify the major fatty acids of 596 lipid species from 33 classes. We estimated narrow-sense heritabilities of lipid species/classes and their genetic correlations with eight CVD traits: BMI, HDL-C, LDL-C, triglycerides, total cholesterol, waist-hip ratio, systolic blood pressure, and diastolic blood pressure. We report heritabilities and genetic correlations of new lipid species/subclasses, including acylcarnitine (AC), ubiquinone, sulfatide, and oxidized cholesteryl esters. Over 99% of lipid species were significantly heritable (h²: 0.06–0.50) and all lipid classes were significantly heritable (h²: 0.14–0.50). The monohexosylceramide and AC classes had the highest median heritabilities (h² = 0.43). The largest genetic correlation was between clinical triglycerides and total diacylglycerol (r_g = 0.88). We observed novel positive genetic correlations between clinical triglycerides and phosphatidylglycerol species (r_g: 0.64–0.82), and HDL-C and alkenylphosphatidylcholine species (r_g: 0.45–0.74). Overall, 51% of the 4,768 lipid species-CVD trait genetic correlations were statistically significant after correction for multiple comparisons. This is the largest lipidomic study to address the heritability of lipids and their genetic correlation with CVD traits. Future work includes identifying putative causal genetic variants for lipid species and CVD using genome-wide SNP and whole-genome sequencing data.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by deleterious mutations in the ABCD1 gene. The ABCD1 protein transports very long-chain FAs (VLCFAs) from the cytosol into the peroxisome where the VLCFAs are degraded through β-oxidation. ABCD1 dysfunction leads to VLCFA accumulation in individuals with X-ALD. FAs are activated by esterification to CoA before metabolic utilization. However, the intracellular pools and metabolic profiles of individual acyl-CoA esters have not been fully analyzed. In this study, we profiled the acyl-CoA species in fibroblasts from X-ALD patients and in ABCD1-deficient HeLa cells. We found that hexacosenoyl (26:1)-CoA, but not hexacosanoyl (26:0)-CoA, was the most abundantly concentrated among the VLCFA-CoA species in these cells. We also show that 26:1-CoA is mainly synthesized from oleoyl-CoA, and the metabolic turnover rate of 26:1-CoA was almost identical to that of oleoyl-CoA in both WT and ABCD1-deficient HeLa cells. The findings of our study provide precise quantitative and metabolic information of each acyl-CoA species in living cells. Our results suggest that VLCFA is endogenously synthesized as VLCFA-CoA through a FA elongation pathway and is then efficiently converted to other metabolites, such as phospholipids, in the absence of ABCD1.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthalmoscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim flash stimuli (<-1.8 log cd·s/m²) occur prior to clinically-recognized diabetic retinopathy. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. Here, we randomized persons with diabetes, without clinically detectable retinopathy, to treatment with either low or high dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control (no DM) subjects. We assessed dim flash stimulated OP delays using a novel hand-held ERG system (RETeval) at baseline, 2 and 4 weeks. RETeval recordings identified significant OP implicit-time delays in persons with diabetes without retinopathy compared to age-matched controls (p<0.001). After two weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a two-week washout. We conclude that detection of dim flash OP delays could provide early detection of DR, and that Sinemet treatment may reverse retinal dysfunction.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Hepatic steatosis, the excess storage of intrahepatic lipids, is a rampant clinical problem associated with the obesity epidemic. Hepatic steatosis is linked to increased risk for insulin resistance, type 2 diabetes, and cardiovascular and advanced liver disease. Accumulating evidence shows that physical activity, exercise, and aerobic capacity have profound effects on regulating intrahepatic lipids and mediating susceptibility for hepatic steatosis. Moreover, exercise can effectively reduce hepatic steatosis independent of changes in body mass. In this perspective, we highlight 1) the relationship between obesity and metabolic pathways putatively driving hepatic steatosis compared with changes induced by exercise; 2) the impact of physical activity, exercise, and aerobic capacity compared with caloric restriction on regulating intrahepatic lipids and steatosis risk; 3) the effects of exercise training (modalities, volume, intensity) for treatment of hepatic steatosis, and 4) evidence for a sustained protection against steatosis induced by exercise. Overall, evidence clearly indicates that exercise powerfully regulates intrahepatic storage of fat and risk for steatosis.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed “templated folding,” whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Cheng Hu
Jan 1, 2018; 67:3-11
Perspectives in Diabetes

6 November 2019

Although some digital platforms now have an impact on more people’s lives than does any one state authority, the international community has been slow to hold to account these platforms’ activities by reference to human rights law. This paper examines how human rights frameworks should guide digital technology.

Mathematical Reviews will be at the JMM in Denver, January 13-18, 2020. The Joint Mathematical Meetings is the largest gathering of mathematicians in the world.  There are lots of great activities:  invited lectures, special sessions, editorial meetings, exhibits, and the chance to … Continue reading →

7 November 2019

The current dispute between the US and China goes far beyond trade tariffs and tit-for-tat reprisals: the underlying driver is a race for global technological supremacy. This paper examines the risks of greater strategic competition as well as potential solutions for mitigating the impacts of the US–China economic confrontation.

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