Publications by authors named "Lori L Jennings"

29 Publications

  • Page 1 of 1

ADAMTSL2 protein and a soluble biomarker signature identify at-risk non-alcoholic steatohepatitis and fibrosis in adults with NAFLD.

J Hepatol 2022 Jan 1;76(1):25-33. Epub 2021 Oct 1.

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

Background & Aims: Identifying fibrosis in non-alcoholic fatty liver disease (NAFLD) is essential to predict liver-related outcomes and guide treatment decisions. A protein-based signature of fibrosis could serve as a valuable, non-invasive diagnostic tool. This study sought to identify circulating proteins associated with fibrosis in NAFLD.

Methods: We used aptamer-based proteomics to measure 4,783 proteins in 2 cohorts (Cohort A and B). Targeted, quantitative assays coupling aptamer-based protein pull down and mass spectrometry (SPMS) validated the profiling results in a bariatric and NAFLD cohort (Cohort C and D, respectively). Generalized linear modeling-logistic regression assessed the ability of candidate proteins to classify fibrosis.

Results: From the multiplex profiling, 16 proteins differed significantly by fibrosis in cohorts A (n = 62) and B (n = 98). Quantitative and robust SPMS assays were developed for 8 proteins and validated in Cohorts C (n = 71) and D (n = 84). The A disintegrin and metalloproteinase with thrombospondin motifs like 2 (ADAMTSL2) protein accurately distinguished non-alcoholic fatty liver (NAFL)/non-alcoholic steatohepatitis (NASH) with fibrosis stage 0-1 (F0-1) from at-risk NASH with fibrosis stage 2-4, with AUROCs of 0.83 and 0.86 in Cohorts C and D, respectively, and from NASH with significant fibrosis (F2-3), with AUROCs of 0.80 and 0.83 in Cohorts C and D, respectively. An 8-protein panel distinguished NAFL/NASH F0-1 from at-risk NASH (AUROCs 0.90 and 0.87 in Cohort C and D, respectively) and NASH F2-3 (AUROCs 0.89 and 0.83 in Cohorts C and D, respectively). The 8-protein panel and ADAMTSL2 protein had superior performance to the NAFLD fibrosis score and fibrosis-4 score.

Conclusion: The ADAMTSL2 protein and an 8-protein soluble biomarker panel are highly associated with at-risk NASH and significant fibrosis; they exhibited superior diagnostic performance compared to standard of care fibrosis scores.

Lay Summary: Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease worldwide. Diagnosing NAFLD and identifying fibrosis (scarring of the liver) currently requires a liver biopsy. Our study identified novel proteins found in the blood which may identify fibrosis without the need for a liver biopsy.
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http://dx.doi.org/10.1016/j.jhep.2021.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8688231PMC
January 2022

Human plasma proteomic profiles indicative of cardiorespiratory fitness.

Nat Metab 2021 06 27;3(6):786-797. Epub 2021 May 27.

Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Maximal oxygen uptake (VOmax) is a direct measure of human cardiorespiratory fitness and is associated with health. However, the molecular determinants of interindividual differences in baseline (intrinsic) VOmax, and of increases of VOmax in response to exercise training (ΔVOmax), are largely unknown. Here, we measure ~5,000 plasma proteins using an affinity-based platform in over 650 sedentary adults before and after a 20-week endurance-exercise intervention and identify 147 proteins and 102 proteins whose plasma levels are associated with baseline VOmax and ΔVOmax, respectively. Addition of a protein biomarker score derived from these proteins to a score based on clinical traits improves the prediction of an individual's ΔVOmax. We validate findings in a separate exercise cohort, further link 21 proteins to incident all-cause mortality in a community-based cohort and reproduce the specificity of ~75% of our key findings using antibody-based assays. Taken together, our data shed light on biological pathways relevant to cardiorespiratory fitness and highlight the potential additive value of protein biomarkers in identifying exercise responsiveness in humans.
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http://dx.doi.org/10.1038/s42255-021-00400-zDOI Listing
June 2021

Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions.

Cell Rep Med 2021 May 3;2(5):100287. Epub 2021 May 3.

Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.
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http://dx.doi.org/10.1016/j.xcrm.2021.100287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091031PMC
May 2021

Effect of longevity genetic variants on the molecular aging rate.

Geroscience 2021 06 4;43(3):1237-1251. Epub 2021 May 4.

Institute for Clinical Research and Health Policy Studies, Tufts Medical Center, Boston, MA, USA.

We conducted a genome-wide association study of 1320 centenarians from the New England Centenarian Study (median age = 104 years) and 2899 unrelated controls using >9 M genetic variants imputed to the HRC panel of ~65,000 haplotypes. The genetic variants with the most significant associations were correlated to 4131 proteins that were profiled in the serum of a subset of 224 study participants using a SOMAscan array. The genetic associations were replicated in a genome-wide association study of 480 centenarians and ~800 controls of Ashkenazi Jewish descent. The proteomic associations were replicated in a proteomic scan of approximately 1000 Ashkenazi Jewish participants from a third cohort. The analysis replicated a protein signature associated with APOE genotypes and confirmed strong overexpression of BIRC2 (p < 5E-16) and under-expression of APOB in carriers of the APOE2 allele (p < 0.05). The analysis also discovered and replicated associations between longevity variants and slower changes of protein biomarkers of aging, including a novel protein signature of rs2184061 (CDKN2A/CDKN2B in chromosome 9) that suggests a genetic regulation of GDF15. The analyses showed that longevity variants correlate with proteome signatures that could be manipulated to discover healthy-aging targets.
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http://dx.doi.org/10.1007/s11357-021-00376-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190315PMC
June 2021

Serum levels of ACE2 are higher in patients with obesity and diabetes.

Obes Sci Pract 2021 Apr 16;7(2):239-243. Epub 2020 Dec 16.

Icelandic Heart Association Kopavogur Iceland.

Objective: As severity of outcome in COVID-19 is disproportionately higher among individuals with obesity, smokers, patients with hypertension, kidney disease, chronic pulmonary disease, coronary heart disease (CHD), and/or type 2 diabetes (T2D), serum levels of ACE2, the cellular entry point for the coronavirus SARS-CoV-2, were examined in these high-risk groups.

Methods: Associations of ACE2 levels to smokers and patients with hypertension, T2D, obesity, CHD, or COPD were investigated in a single center population-based study of 5457 Icelanders from the Age, Gene/Environment Susceptibility Reykjavík Study (AGES-RS) of the elderly (mean age 75 ± 6 years), using multiple linear regression analysis.

Results: Serum levels of ACE2 were higher in smokers and individuals with T2D and/or obesity while they were unaffected in the other patient groups.

Conclusion: ACE2 levels are higher in some patient groups with comorbidities linked to COVID-19 including obesity and T2D and as such may have an emerging role as a circulating biomarker for severity of outcome in the disease.
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http://dx.doi.org/10.1002/osp4.472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019273PMC
April 2021

BET bromodomain inhibitors regulate keratinocyte plasticity.

Nat Chem Biol 2021 03 18;17(3):280-290. Epub 2021 Jan 18.

Novartis Institutes for BioMedical Research, Basel, Switzerland.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
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http://dx.doi.org/10.1038/s41589-020-00716-zDOI Listing
March 2021

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients.

bioRxiv 2020 Nov 3. Epub 2020 Nov 3.

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.
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http://dx.doi.org/10.1101/2020.11.02.365536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654866PMC
November 2020

It's in Our Blood: A Glimpse of Personalized Medicine.

Trends Mol Med 2021 01 25;27(1):20-30. Epub 2020 Sep 25.

Icelandic Heart Association, IS-201 Kopavogur, Iceland; Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. Electronic address:

Recent advances in protein profiling technology has facilitated simultaneous measurement of thousands of proteins in large population studies, exposing the depth and complexity of the plasma and serum proteomes. This revealed that proteins in circulation were organized into regulatory modules under genetic control and closely associated with current and future common diseases. Unlike networks in solid tissues, serum protein networks comprise members synthesized across different tissues of the body. Genetic analysis reveals that this cross-tissue regulation of the serum proteome participates in systemic homeostasis and mirrors the global disease state of individuals. Here, we discuss how application of this information in routine clinical evaluations may transform the future practice of medicine.
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http://dx.doi.org/10.1016/j.molmed.2020.09.003DOI Listing
January 2021

ACE2 levels are altered in comorbidities linked to severe outcome in COVID-19.

medRxiv 2020 Jun 5. Epub 2020 Jun 5.

Aims: Severity of outcome in COVID-19 is disproportionately higher among the obese, males, smokers, those suffering from hypertension, kidney disease, coronary heart disease (CHD) and/or type 2 diabetes (T2D). We examined if serum levels of ACE2, the cellular entry point for the coronavirus SARS-CoV-2, were altered in these high-risk groups.

Methods: Associations of serum ACE2 levels to hypertension, T2D, obesity, CHD, smokers and males in a single center population-based study of 5457 Icelanders from the Age, Gene/Environment Susceptibility Reykjavik Study (AGES-RS) of the elderly (mean age 75+/-6 years).

Results: Smokers, males, and individuals with T2D or obesity have altered serum levels of ACE2 that may influence productive infection of SARS-CoV-2 in these high-risk groups.

Conclusion: ACE2 levels are upregulated in some patient groups with comorbidities linked to COVID-19 and as such may have an emerging role as a circulating biomarker for severity of outcome in COVID-19.
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http://dx.doi.org/10.1101/2020.06.04.20122044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276056PMC
June 2020

Antihypertensive medication uses and serum ACE2 levels: ACEIs/ARBs treatment does not raise serum levels of ACE2.

medRxiv 2020 May 25. Epub 2020 May 25.

Icelandic Heart Association, Holtasmari 1, IS-201 Kopavogur, Iceland.

Importance: Recent reports have shown that hypertension is the most common comorbidity associated with mortality in the current coronavirus disease 2019 (COVID-19). This has been related to the use of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) as animal studies indicate that these medications increase levels of ACE2, the cellular entry point for the coronavirus SARS-CoV-2. This has prompted clinicians to recommend discontinuing ACEIs and ARBs.

Objective: To examine the effect of ACEIs or ARBs treatment on serum levels of ACE2 and other key enzymes in the renin-angiotensin system (RAS).

Design Setting And Participants: A single center population-based study of 5457 Icelanders from the Age, Gene/Environment Susceptibility Reykjavik Study (AGES-RS) of the elderly (mean age 75±6 years) stratified by ACEIs (N = 699) or ARBs (N = 753) treatment.

Main Outcomes And Measures: The AGES-RS study population was stratified by ACEIs and ARBs medication use and compared for age, body mass index (BMI) (kg/m), hypertension and type 2 diabetes (T2D) as well as serum levels of renin, ACE and ACE2.

Results: While renin and ACE levels were significantly raised in serum of individuals on ACEIs or ARBs treatments, the ACE2 levels remained unaffected.

Conclusions And Relevance: Treatment with ACEIs or ARBs does not raise ACE2 levels in serum. Therefore, the present study does not support the proposed discontinuation of these medications among patients affected with COVID-19.
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http://dx.doi.org/10.1101/2020.05.21.20108738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265694PMC
May 2020

Circulating Protein Signatures and Causal Candidates for Type 2 Diabetes.

Diabetes 2020 08 8;69(8):1843-1853. Epub 2020 May 8.

Faculty of Medicine, University of Iceland, Reykjavik, Iceland

The increasing prevalence of type 2 diabetes poses a major challenge to societies worldwide. Blood-based factors like serum proteins are in contact with every organ in the body to mediate global homeostasis and may thus directly regulate complex processes such as aging and the development of common chronic diseases. We applied a data-driven proteomics approach, measuring serum levels of 4,137 proteins in 5,438 elderly Icelanders, and identified 536 proteins associated with prevalent and/or incident type 2 diabetes. We validated a subset of the observed associations in an independent case-control study of type 2 diabetes. These protein associations provide novel biological insights into the molecular mechanisms that are dysregulated prior to and following the onset of type 2 diabetes and can be detected in serum. A bidirectional two-sample Mendelian randomization analysis indicated that serum changes of at least 23 proteins are downstream of the disease or its genetic liability, while 15 proteins were supported as having a causal role in type 2 diabetes.
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http://dx.doi.org/10.2337/db19-1070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372075PMC
August 2020

Predicting health and life span with the deep plasma proteome.

Nat Med 2019 12;25(12):1815-1816

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41591-019-0677-yDOI Listing
December 2019

Oncostatin M reduces atherosclerosis development in APOE*3Leiden.CETP mice and is associated with increased survival probability in humans.

PLoS One 2019 28;14(8):e0221477. Epub 2019 Aug 28.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Objective: Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied.

Approach And Results: Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457).

Conclusions: Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221477PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713386PMC
March 2020

A serum protein signature of APOE genotypes in centenarians.

Aging Cell 2019 12 5;18(6):e13023. Epub 2019 Aug 5.

Geriatrics Section, Department of Medicine, School of Medicine and Boston Medical Center, Boston University, Boston, MA.

The discovery of treatments to prevent or delay dementia and Alzheimer's disease is a priority. The gene APOE is associated with cognitive change and late-onset Alzheimer's disease, and epidemiological studies have provided strong evidence that the e allele of APOE has a neuroprotective effect, it is associated with increased longevity and an extended healthy lifespan in centenarians. In this study, we correlated APOE genotype data of 222 participants of the New England Centenarian Study, including 75 centenarians, 82 centenarian offspring, and 65 controls, comprising 55 carriers of APOE e , with aptamer-based serum proteomics (SomaLogic technology) of 4,785 human proteins corresponding to 4,137 genes. We discovered a signature of 16 proteins that associated with different APOE genotypes and replicated the signature in three independent studies. We also show that the protein signature tracks with gene expression profiles in brains of late-onset Alzheimer's disease versus healthy controls. Finally, we show that seven of these proteins correlate with cognitive function patterns in longitudinally collected data. This analysis in particular suggests that Baculoviral IAP repeat containing two (BIRC2) is a novel biomarker of neuroprotection that associates with the neuroprotective allele of APOE. Therefore, targeting APOE e molecularly may preserve cognitive function.
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http://dx.doi.org/10.1111/acel.13023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826130PMC
December 2019

Co-regulatory networks of human serum proteins link genetics to disease.

Science 2018 08 2;361(6404):769-773. Epub 2018 Aug 2.

Icelandic Heart Association, Holtasmari 1, IS-201 Kopavogur, Iceland.

Proteins circulating in the blood are critical for age-related disease processes; however, the serum proteome has remained largely unexplored. To this end, 4137 proteins covering most predicted extracellular proteins were measured in the serum of 5457 Icelanders over 65 years of age. Pairwise correlation between proteins as they varied across individuals revealed 27 different network modules of serum proteins, many of which were associated with cardiovascular and metabolic disease states, as well as overall survival. The protein modules were controlled by cis- and trans-acting genetic variants, which in many cases were also associated with complex disease. This revealed co-regulated groups of circulating proteins that incorporated regulatory control between tissues and demonstrated close relationships to past, current, and future disease states.
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http://dx.doi.org/10.1126/science.aaq1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190714PMC
August 2018

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions.

Circulation 2018 03 8;137(12):1270-1277. Epub 2017 Dec 8.

Cardiovascular Research Center (D.N., M.J.K., L.A.F., R.E.G.)

Background: Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury.

Methods: We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls.

Results: We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; <1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; <1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; <6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers.

Conclusions: Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.029443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860961PMC
March 2018

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Cell Metab 2015 Jul 19;22(1):164-74. Epub 2015 May 19.

Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA. Electronic address:

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-β molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.
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http://dx.doi.org/10.1016/j.cmet.2015.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497834PMC
July 2015

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.

Proc Natl Acad Sci U S A 2010 Feb 2;107(8):3552-7. Epub 2010 Feb 2.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFbeta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.
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http://dx.doi.org/10.1073/pnas.0914019107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840467PMC
February 2010

Fragmented tissue factor pathway inhibitor (TFPI) and TFPI C-terminal peptides eliminate serum-resistant Escherichia coli from blood cultures.

J Infect Dis 2009 Jun;199(12):1807-15

Novartis Institutes for Biomedical Research, Emeryville, California 94608, USA.

Background: Tissue factor pathway inhibitor (TFPI) is a major regulator of blood clotting. Receipt of recombinant TFPI (rTFPI) protected animals from death in Escherichia coli-induced severe sepsis models and is under evaluation in a phase III clinical trial involving patients with severe community-acquired pneumonia. Because the mechanism of action of rTFPI in acute bacterial infection is not well understood, we sought to identify and map rTFPI peptides that have antimicrobial activity against E. coli.

Methods: Fragmented rTFPI and C-terminal TFPI peptide activities against pathogenic E. coli strains were measured in ex vivo blood cultures and in serum.

Results: The C-terminal peptides exhibited complement-dependent antibacterial activity and directly interacted with the bacterial cell surface of E. coli. Both complement-mediated killing and cell-surface binding were reversed by low amounts of heparin.

Conclusions: Our investigation revealed a previously unidentified mechanism of antibacterial activity for TFPI. C-terminal rTFPI fragments kill serum-resistant E. coli though the complement pathway of the innate immune system, suggesting a multimodal mechanism of action of rTFPI that may assist in reducing mortality in animal models of severe sepsis and contribute to therapeutic effectiveness.
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http://dx.doi.org/10.1086/599097DOI Listing
June 2009

Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins.

Biochemistry 2006 Oct;45(42):12816-27

Department of Pharmacology, University of California-San Diego, La Jolla, California 92093-0636, USA.

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.
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http://dx.doi.org/10.1021/bi0614131DOI Listing
October 2006

A single mutation near the C-terminus in alpha/beta hydrolase fold protein family causes a defect in protein processing.

Chem Biol Interact 2005 Dec;157-158:371-2

Department of Pharmacology and National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA 92093-0636, USA.

An Arg to Cys mutation in the extracellular domain of neuroligin-3 (NL3) was recently found in a twin set with autism [S. Jamain, H. Quach, C. Betancur, M. Rastam, C. Colineaux, I.C. Gillberg, H. Soderstrom, B. Giros, M. Leboyer, C. Gillberg, T. Bourgeron, Paris Autism Research International Sibpair Study, mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism, Nat. Genet. 34 (2003) 27-29]. The Cys substitution in NL3 causes altered intracellular protein trafficking, intracellular retention and diminished association with its cognate partner, beta-neurexin [D. Comoletti, A. De Jaco, L.L. Jennings, R.E. Flynn, G. Gaietta, I. Tsigelny, M.H. Ellisman, P. Taylor, The R451C-neuroligin-3 mutation associated with autism reveals a defect in protein processing, J. Neurosci. 24 (2004) 4889-4893]. NL3, butyrylcholinesterase (BuChE), and acetylcholinesterase (AChE), as members of the (/(-hydrolase fold family of proteins, share over 30% of amino acid identity in their extracellular domains. In particular, Arg451 in NL3 is conserved in the alpha/beta-hydrolase fold family being homologous to Arg386 in BuChE and Arg395 in AChE. A Cys substitution at the homologous Arg in the BuChE was found studying post-succinylcholine apnea in an Australian population [T. Yen, B.N. Nightingale, J.C. Burns, D.R. Sullivan, P.M. Stewart, Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population, Clin. Chem. 49 (2003) 1297-308]. We have made the homologous mutation in the mouse AChE and BuChE genes and showed that the Arg to Cys mutations resulted in identical alterations in the cellular phenotype for the various members of the alpha/beta-hydrolase fold family proteins.
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http://dx.doi.org/10.1016/j.cbi.2005.10.057DOI Listing
December 2005

The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in protein processing.

J Neurosci 2004 May;24(20):4889-93

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA.

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the beta-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished beta-neurexin-1 (NX1beta) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1beta. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.0468-04.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729460PMC
May 2004

Structural characterization of recombinant soluble rat neuroligin 1: mapping of secondary structure and glycosylation by mass spectrometry.

Biochemistry 2004 Feb;43(6):1496-506

Department of Pharmacology and Howard Hughes Medical Institute Mass Spectrometry Facility, University of California, San Diego, La Jolla 92093, USA.

Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microheterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NL1 established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.
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http://dx.doi.org/10.1021/bi035278tDOI Listing
February 2004

Characterization of the interaction of a recombinant soluble neuroligin-1 with neurexin-1beta.

J Biol Chem 2003 Dec 30;278(50):50497-505. Epub 2003 Sep 30.

Department of Pharmacology, University of California, La Jolla, California 92093-0636, USA.

Neuroligins, proteins of the alpha/beta-hydrolase fold family, are found as postsynaptic transmembrane proteins whose extracellular domain associates with presynaptic partners, proteins of the neurexin family. To characterize the molecular basis of neuroligin interaction with neurexin-beta, we expressed five soluble and exportable forms of neuroligin-1 from recombinant DNA sources, by truncating the protein before the transmembrane span near its carboxyl terminus. The extracellular domain of functional neuroligin-1 associates as a dimer when analyzed by sedimentation equilibrium. By surface plasmon resonance, we established that soluble neuroligins-1 bind neurexin-1beta, but the homologous alpha/beta-hydrolase fold protein, acetylcholinesterase, failed to associate with the neurexins. Neuroligin-1 has a unique N-linked glycosylation pattern in the neuroligin family, and glycosylation and its processing modify neuroligin activity. Incomplete processing of the protein and enzymatic removal of the oligosaccharides chain or the terminal sialic acids from neuroligin-1 enhance its activity, whereas deglycosylation of neurexin-1beta did not alter its association capacity. In particular, the N-linked glycosylation at position 303 appears to be a major determinant in modifying the association with neurexin-1beta. We show here that glycosylation processing of neuroligin, in addition to mRNA splicing and gene selection, contributes to the specificity of the neurexin-beta/neuroligin-1 association.
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http://dx.doi.org/10.1074/jbc.M306803200DOI Listing
December 2003

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry.

Biochemistry 2003 Sep;42(37):11083-91

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.
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http://dx.doi.org/10.1021/bi034756xDOI Listing
September 2003

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate.

J Biol Chem 2003 Nov 21;278(46):45512-8. Epub 2003 Aug 21.

Center for Environmental Health Sciences, The University of Montana, Missoula, Montana 59812, USA.

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.
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http://dx.doi.org/10.1074/jbc.M304781200DOI Listing
November 2003

Immunohistochemical variation of human equilibrative nucleoside transporter 1 protein in primary breast cancers.

Clin Cancer Res 2002 Jan;8(1):110-6

Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2 Canada.

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.
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January 2002
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