Publications by authors named "Richard Leduc"

151 Publications

Inhibitors of type II transmembrane serine proteases in the treatment of diseases of the respiratory tract - A review of patent literature.

Expert Opin Ther Pat 2020 Nov 12;30(11):807-824. Epub 2020 Oct 12.

Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke , Sherbrooke (Québec), Canada.

Introduction: Type II transmembrane serine proteases (TTSPs) of the human respiratory tract generate high interest owing to their ability, among other roles, to cleave surface proteins of respiratory viruses. This step is critical in the viral invasion of coronaviruses, including SARS-CoV-2 responsible for COVID-19, but also influenza viruses and reoviruses. Accordingly, these cell surface enzymes constitute appealing therapeutic targets to develop host-based therapeutics against respiratory viral diseases. Additionally, their deregulated levels or activity has been described in non-viral diseases such as fibrosis, cancer, and osteoarthritis, making them potential targets in these indications.

Areas Covered: Areas covered: This review includes WIPO-listed patents reporting small molecules and peptide-based inhibitors of type II transmembrane serine proteases of the respiratory tract.

Expert Opinion: Expert opinion: Several TTSPs of the respiratory tract represent attractive pharmacological targets in the treatment of respiratory infectious diseases (notably COVID-19 and influenza), but also against idiopathic pulmonary fibrosis and lung cancer. The current emphasis is primarily on TMPRSS2, matriptase, and hepsin, yet other TTSPs await validation. Compounds listed herein are predominantly peptidomimetic inhibitors, some with covalent reversible mechanisms of action and high potencies. Their selectivity profile, however, are often only partially characterized. Preclinical data are promising and warrant further advancement in the above diseases.
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http://dx.doi.org/10.1080/13543776.2020.1817390DOI Listing
November 2020

Matriptase processing of APLP1 ectodomain alters its homodimerization.

Sci Rep 2020 06 22;10(1):10091. Epub 2020 Jun 22.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, J1H5N4, Canada.

The amyloid beta peptide (Aβ) is derived from the amyloid precursor protein (APP) by secretase processing. APP is also cleaved by numerous other proteases, such as the type II transmembrane serine protease matriptase, with consequences on the production of Aβ. Because the APP homolog protein amyloid-like protein 1 (APLP1) shares similarities with APP, we sought to determine if matriptase also plays a role in its processing. Here, we demonstrate that matriptase directly interacts with APLP1 and that APLP1 is cleaved in cellulo by matriptase in its E1 ectodomains at arginine 124. Replacing Arg124 with Ala abolished APLP1 processing by matriptase. Using a bioluminescence resonance energy transfer (BRET) assay we found that matriptase reduces APLP1 homodimeric interactions. This study identifies matriptase as the first protease cleaving APLP1 in its dimerization domain, potentially altering the multiple functions associated with dimer formation.
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http://dx.doi.org/10.1038/s41598-020-67005-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308337PMC
June 2020

Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics.

J Am Soc Mass Spectrom 2020 Jul 24;31(7):1398-1409. Epub 2020 Jun 24.

Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, United States.

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.
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http://dx.doi.org/10.1021/jasms.0c00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539637PMC
July 2020

Development of Novel -In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor Overexpressed in Solid Tumours.

Biomolecules 2020 03 19;10(3). Epub 2020 Mar 19.

Institute for Research and Innovation in Biomedicine (IRIB), University of Rouen Normandy, INSERM U1239, DC2N, Rouen, France, 76000 Rouen, France.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. In-DOTA-hUII or In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.
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http://dx.doi.org/10.3390/biom10030471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175314PMC
March 2020

Proteomic analyses of decellularized porcine ovaries identified new matrisome proteins and spatial differences across and within ovarian compartments.

Sci Rep 2019 12 27;9(1):20001. Epub 2019 Dec 27.

Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, USA.

Premature ovarian insufficiency (POI) affects approximately 1% of women. We aim to understand the ovarian microenvironment, including the extracellular matrix (ECM) and associated proteins (matrisome), and its role in controlling folliculogenesis. We mapped the composition of the matrisome of porcine ovaries through the cortical compartment, where quiescent follicles reside and the medullary compartment, where the larger follicles grow and mature. To do this we sliced the ovaries, uniformly in two anatomical planes, enriched for matrisome proteins and performed bottom-up shotgun proteomic analyses. We identified 42 matrisome proteins that were significantly differentially expressed across depths, and 11 matrisome proteins that have not been identified in previous ovarian protein analyses. We validated these data for nine proteins and confirmed compartmental differences with a second processing method. Here we describe a processing and proteomic analysis pipeline that revealed spatial differences and matrisome protein candidates that may influence folliculogenesis.
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http://dx.doi.org/10.1038/s41598-019-56454-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934504PMC
December 2019

Multidimensional Top-Down Proteomics of Brain-Region-Specific Mouse Brain Proteoforms Responsive to Cocaine and Estradiol.

J Proteome Res 2019 11 2;18(11):3999-4012. Epub 2019 Oct 2.

Department of Psychiatry , University of Illinois at Chicago , 1601 West Taylor Street , Chicago , Illinois 60612 , United States.

Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17β-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.
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http://dx.doi.org/10.1021/acs.jproteome.9b00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917473PMC
November 2019

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Cell Chem Biol 2019 11 19;26(11):1559-1572.e9. Epub 2019 Sep 19.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12(e) Avenue Nord, Sherbrooke, QC J1H 5N4, Canada; Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address:

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.
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http://dx.doi.org/10.1016/j.chembiol.2019.09.004DOI Listing
November 2019

Interplay between intracellular loop 1 and helix VIII of the angiotensin II type 2 receptor controls its activation.

Biochem Pharmacol 2019 10 23;168:330-338. Epub 2019 Jul 23.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada; Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada. Electronic address:

The signaling mechanisms of the angiotensin II type 2 receptor (ATR), a heptahelical receptor, have not yet been clearly and completely defined. In the present contribution, we set out to identify the molecular determinants involved in ATR activation. Although ATR has not been shown to engage G, G, G, and β-arrestin (βarr) pathways as does the ATR upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published ATR structure may play a role in the receptor's capacity to couple to downstream effectors. In the ATR structure, helix VIII points inwards and towards intracellular loop 3 (ICL3) to form tertiary interactions with transmembrane domain 6 (TM6), possibly impeding access to signaling effectors. On the other hand, in most class A GPCRs, helix VIII is found to be engaged in tertiary interactions with ICL1 and away from the effector binding site. Upon closer examination of the ATR structure, we found that the residues contained within intracellular loop 1 (ICL1) may be involved in driving this unusual conformation of helix VIII. To explore this hypothesis, we designed a series of ATR/ATR receptor chimeras to validate the roles of ICL1 and helix VIII in ATR signaling. Substituting the ATR ICL1 into ATR led to a mutant receptor that coupled to G. The substitution of the helix VIII and C-terminal domains of ATR into the ATR backbone led to a mutant receptor that retained ATR-like signaling properties. These results suggest that the C-terminal portion of ATR is compatible with canonical GPCR signaling and that ICL1 of ATR is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or βarrs.
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http://dx.doi.org/10.1016/j.bcp.2019.07.018DOI Listing
October 2019

Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding.

J Biol Chem 2019 08 27;294(33):12459-12471. Epub 2019 Jun 27.

Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, Florida 32608. Electronic address:

NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.
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http://dx.doi.org/10.1074/jbc.RA118.006159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699848PMC
August 2019

Identification and Quantification of Proteoforms by Mass Spectrometry.

Proteomics 2019 05;19(10):e1800361

Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.
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http://dx.doi.org/10.1002/pmic.201800361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602557PMC
May 2019

Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics.

Mol Cell Proteomics 2019 04 15;18(4):796-805. Epub 2019 Jan 15.

From the ‡Proteomics Center of Excellence, Northwestern University, Evanston, Illinois;; §Department of Molecular Biosciences, Northwestern University, Evanston, Illinois;; Department of Chemistry and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois. Electronic address:

Within the last several years, top-down proteomics has emerged as a high throughput technique for protein and proteoform identification. This technique has the potential to identify and characterize thousands of proteoforms within a single study, but the absence of accurate false discovery rate (FDR) estimation could hinder the adoption and consistency of top-down proteomics in the future. In automated identification and characterization of proteoforms, FDR calculation strongly depends on the context of the search. The context includes MS data quality, the database being interrogated, the search engine, and the parameters of the search. Particular to top-down proteomics-there are four molecular levels of study: proteoform spectral match (PrSM), protein, isoform, and proteoform. Here, a context-dependent framework for calculating an accurate FDR at each level was designed, implemented, and validated against a manually curated training set with 546 confirmed proteoforms. We examined several search contexts and found that an FDR calculated at the PrSM level under-reported the true FDR at the protein level by an average of 24-fold. We present a new open-source tool, the TDCD_FDR_Calculator, which provides a scalable, context-dependent FDR calculation that can be applied post-search to enhance the quality of results in top-down proteomics from any search engine.
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http://dx.doi.org/10.1074/mcp.RA118.000993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442365PMC
April 2019

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.

Sci Signal 2018 12 4;11(559). Epub 2018 Dec 4.

Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, QC H4A 3J1, Canada.

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gα and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.
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http://dx.doi.org/10.1126/scisignal.aat1631DOI Listing
December 2018

Functional diversity of TMPRSS6 isoforms and variants expressed in hepatocellular carcinoma cell lines.

Sci Rep 2018 08 22;8(1):12562. Epub 2018 Aug 22.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.

TMPRSS6, also known as matriptase-2, is a type II transmembrane serine protease that plays a major role in iron homeostasis by acting as a negative regulator of hepcidin production through cleavage of the BMP co-receptor haemojuvelin. Iron-refractory iron deficiency anaemia (IRIDA), an iron metabolism disorder, is associated with mutations in the TMPRSS6 gene. By analysing RNA-seq data encoding TMPRSS6 isoforms and other proteins involved in hepcidin production, we uncovered significant differences in expression levels between hepatocellular carcinoma (HCC) cell lines and normal human liver samples. Most notably, TMPRSS6 and HAMP expression was found to be much lower in HepG2 and Huh7 cells when compared to human liver samples. Furthermore, we characterized the common TMPRSS6 polymorphism V736A identified in Hep3B cells, the V795I mutation found in HepG2 cells, also associated with IRIDA, and the G603R substitution recently detected in two IRIDA patients. While variant V736A is as active as wild-type TMPRSS6, mutants V795I and G603R displayed significantly reduced proteolytic activity. Our results provide important information about commonly used liver cell models and shed light on the impact of two TMPRSS6 mutations associated with IRIDA.
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http://dx.doi.org/10.1038/s41598-018-30618-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105633PMC
August 2018

GRK2 knockdown in mice exacerbates kidney injury and alters renal mechanisms of blood pressure regulation.

Sci Rep 2018 07 30;8(1):11415. Epub 2018 Jul 30.

University of Ottawa Brain and Mind Research Institute and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.

The renin-angiotensin system regulates blood pressure and fluid balance in the body primarily via angiotensin receptor 1 (AT1R). Renal AT1R was found to be primarily responsible for Ang II-mediated hypertension. G protein-coupled receptor kinase 2 (GRK2) modulates AT1R desensitization and increased GRK2 protein expression is reported in hypertensive patients. However, the consequences of GRK2 inhibition on kidney functions remain unknown. We employed shGRK2 knockdown mice (shGRK2 mice) to test the role of GRK2 in kidney development and function that can be ultimately linked to the hypertensive phenotype detected in shGRK2 mice. GRK2 knockdown reduced kidney size, nephrogenesis and glomerular count, and impaired glomerular filtration. Glomerular damage in adult shGRK2 mice was associated with increased renin- and AT1R-mediated production of reactive oxygen species. The AT1R blocker, Losartan, normalized elevated blood pressure and markedly improved glomerular filtration in the shGRK2 knockdown mice. Our findings provide evidence for the crucial role of GRK2 in renal regulation of blood pressure. It also suggests that the detrimental outcomes of GRK2 inhibitors on the kidney should be carefully examined when used as antihypertensive.
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http://dx.doi.org/10.1038/s41598-018-29876-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065385PMC
July 2018

Structural Optimization and Characterization of Potent Analgesic Macrocyclic Analogues of Neurotensin (8-13).

J Med Chem 2018 08 13;61(16):7103-7115. Epub 2018 Aug 13.

Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke , Université de Sherbrooke , 3001 12e Avenue Nord , Sherbrooke , Quebec J1H 5N4 , Canada.

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( K improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00175DOI Listing
August 2018

Label-free cell signaling pathway deconvolution of angiotensin type 1 receptor reveals time-resolved G-protein activity and distinct AngII and AngIIIIV responses.

Pharmacol Res 2018 10 28;136:108-120. Epub 2018 Jun 28.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, J1H5N4, Canada; Institut de pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, J1H5N4, Canada. Electronic address:

Angiotensin II (AngII) type 1 receptor (ATR) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. ATR has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human ATR. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of ATR. First, we assessed the contributions of Gq, G12/13, Gi, Gβγ, ERK1/2 and β-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with β-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the ATR-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous ATR ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested ATR biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties.
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http://dx.doi.org/10.1016/j.phrs.2018.06.027DOI Listing
October 2018

The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins.

Anal Chem 2018 07 5;90(14):8553-8560. Epub 2018 Jul 5.

Morgridge Institute for Research , Madison , Wisconsin 53706 , United States.

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.
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http://dx.doi.org/10.1021/acs.analchem.8b01638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050102PMC
July 2018

Angiotensin II cyclic analogs as tools to investigate ATR biased signaling mechanisms.

Biochem Pharmacol 2018 08 20;154:104-117. Epub 2018 Apr 20.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada. Electronic address:

G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (ATR). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([SarHcy]AngII, [SarCysHcy]AngII and [SarCys]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and β-arrestin (βarr) signaling pathways via ATR. Interestingly, [SarHcy]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe of AngII within the hydrophobic core of ATR, associated with Gq/11 activation, is increased with [SarHcy]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism.
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http://dx.doi.org/10.1016/j.bcp.2018.04.021DOI Listing
August 2018

Evaluation of PM surface concentrations simulated by Version 1 of NASA's MERRA Aerosol Reanalysis over Europe.

Atmos Pollut Res 2017 Mar 3;8(2):374-382. Epub 2016 Nov 3.

Département de géographie, Université Laval, Quebec City, Quebec, Canada.

This article evaluates the concentrations of particulate matter (PM) and some of its chemical speciation such as sulfate, organic carbon, black carbon and sea salt particles simulated at the surface by Version 1 of the Aerosol Reanalysis of NASA's Modern-Era Retrospective Analysis for Research and Application (MERRAero) over Europe. Measurement data from the European Monitoring and Evaluation Programme database were used. The concentrations of coarse PM (PM), fine PM (PM), sulfate and black carbon particles are overall well simulated, despite a slight and consistent overestimation of PM concentration, and a slight and consistent underestimation of PM and sulfate concentrations throughout most of the year. The concentration of organic carbon was largely underestimated, especially in winter, caused by two specific monitoring stations in Italy, resulting in an overall poor performance for this particular species. After removing these two stations from the sample, the evaluation of OC substantially improved but an underestimation in winter remained. Carbon emissions originating from anthropogenic sources, such as residential wood burning in winter, unresolved by MERRAero provide a plausible explanation for this discrepancy.. The evaluation of PM, sulfate and organic carbon concentrations improved during the summer. The concentration of fine sea salt particles was consistently and largely overestimated, but contributes relatively little to total PM concentration.
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http://dx.doi.org/10.1016/j.apr.2016.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887125PMC
March 2017

Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.

Proc Natl Acad Sci U S A 2018 04 2;115(16):4140-4145. Epub 2018 Apr 2.

Department of Chemistry, Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208;

Mutations of the gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.
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http://dx.doi.org/10.1073/pnas.1716122115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910823PMC
April 2018

In Search of the Optimal Macrocyclization Site for Neurotensin.

ACS Med Chem Lett 2018 Mar 29;9(3):227-232. Epub 2018 Jan 29.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec J1H 5N4, Canada.

Neurotensin exerts potent analgesic effects following activation of its cognate GPCRs. In this study, we describe a systematic exploration, using structure-based design, of conformationally constraining neurotensin (8-13) with the help of macrocyclization and the resulting impacts on binding affinity, signaling, and proteolytic stability. This exploratory study led to a macrocyclic scaffold with submicromolar binding affinity, agonist activity, and greatly improved plasma stability.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846049PMC
March 2018

The hypotensive effect of activated apelin receptor is correlated with β-arrestin recruitment.

Pharmacol Res 2018 05 9;131:7-16. Epub 2018 Mar 9.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada; Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Electronic address:

The apelinergic system is an important player in the regulation of both vascular tone and cardiovascular function, making this physiological system an attractive target for drug development for hypertension, heart failure and ischemic heart disease. Indeed, apelin exerts a positive inotropic effect in humans whilst reducing peripheral vascular resistance. In this study, we investigated the signaling pathways through which apelin exerts its hypotensive action. We synthesized a series of apelin-13 analogs whereby the C-terminal Phe residue was replaced by natural or unnatural amino acids. In HEK293 cells expressing APJ, we evaluated the relative efficacy of these compounds to activate Gα and Gα G-proteins, recruit β-arrestins 1 and 2 (βarrs), and inhibit cAMP production. Calculating the transduction ratio for each pathway allowed us to identify several analogs with distinct signaling profiles. Furthermore, we found that these analogs delivered i.v. to Sprague-Dawley rats exerted a wide range of hypotensive responses. Indeed, two compounds lost their ability to lower blood pressure, while other analogs significantly reduced blood pressure as apelin-13. Interestingly, analogs that did not lower blood pressure were less effective at recruiting βarrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with βarr recruitment but not with G protein-dependent signaling. In conclusion, our results demonstrated that the βarr recruitment potency is involved in the hypotensive efficacy of activated APJ.
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http://dx.doi.org/10.1016/j.phrs.2018.02.032DOI Listing
May 2018

Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.

Anal Chem 2018 03 26;90(6):3802-3810. Epub 2018 Feb 26.

Departments of Chemistry, Molecular Biosciences and the Proteomics Center of Excellence , Northwestern University , 2145 North Sheridan Road , Evanston , Illinois 60208 , United States.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.
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http://dx.doi.org/10.1021/acs.analchem.7b04108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861018PMC
March 2018

Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities.

J Cell Mol Med 2018 04 14;22(4):2498-2509. Epub 2018 Feb 14.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.

TMPRSS6 (matriptase-2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA-seq data and RT-PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6-1) and 3 are mostly expressed in human testis while TMPRSS6-2 and TMPRSS6-4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6-1 undergoes internalization. Moreover, truncated TMPRSS6-3 or catalytically altered TMPRSS6-4 interact with HJV and prevent its cleavage by TMPRSS6-2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.
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http://dx.doi.org/10.1111/jcmm.13562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867103PMC
April 2018

ProForma: A Standard Proteoform Notation.

J Proteome Res 2018 03 14;17(3):1321-1325. Epub 2018 Feb 14.

Spectroswiss , 1015 Lausanne, Switzerland.

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.
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http://dx.doi.org/10.1021/acs.jproteome.7b00851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837035PMC
March 2018

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover.

Sci Rep 2017 12 1;7(1):16693. Epub 2017 Dec 1.

Skeletal Research Group, Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK.

Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.
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http://dx.doi.org/10.1038/s41598-017-17028-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711915PMC
December 2017

Gα protein binds ubiquitin to regulate epidermal growth factor receptor endosomal sorting.

Proc Natl Acad Sci U S A 2017 12 30;114(51):13477-13482. Epub 2017 Nov 30.

Department of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4;

The Gα subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gα in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gα in this sorting step is still poorly characterized. Here, we report that Gα interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gα contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in Gα confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gα as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gα in receptor trafficking and signaling for the fine-tuning of the cellular response.
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http://dx.doi.org/10.1073/pnas.1708215114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754765PMC
December 2017

The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to β-amyloid peptide.

J Biol Chem 2017 12 20;292(50):20669-20682. Epub 2017 Oct 20.

From the Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4, Canada,

Recent studies have reported that many proteases, besides the canonical α-, β-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate β-amyloid (Aβ) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aβ40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aβ and in Alzheimer's disease pathology.
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http://dx.doi.org/10.1074/jbc.M117.792911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733603PMC
December 2017

Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain.

Sci Rep 2017 08 25;7(1):9520. Epub 2017 Aug 25.

Department of Neurology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, 63110, United States.

Amyloid-beta (Aβ) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aβ from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aβ. We found a diversity of Aβ peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aβ proteoforms detected. The Aβ proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aβ comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aβ proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aβ proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.
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http://dx.doi.org/10.1038/s41598-017-10422-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572664PMC
August 2017