Publications by authors named "Àlex Bayés"

29 Publications

  • Page 1 of 1

AMPA receptor auxiliary subunits emerged during early vertebrate evolution by neo/subfunctionalization of unrelated proteins.

Open Biol 2020 10 28;10(10):200234. Epub 2020 Oct 28.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, Barcelona, Spain.

In mammalian synapses, the function of ionotropic glutamate receptors is critically modulated by auxiliary subunits. Most of these specifically regulate the synaptic localization and electrophysiological properties of AMPA-type glutamate receptors (AMPARs). Here, we comprehensively investigated the animal evolution of the protein families that contain AMPAR auxiliary subunits (ARASs). We observed that, on average, vertebrates have four times more ARASs than other animal species. We also demonstrated that ARASs belong to four unrelated protein families: CACNG-GSG1, cornichon, shisa and Dispanin C. Our study demonstrates that, despite the ancient origin of these four protein families, the majority of ARASs emerged during vertebrate evolution by independent but convergent processes of neo/subfunctionalization that resulted in the multiple ARASs found in present vertebrate genomes. Importantly, although AMPARs appeared and diversified in the ancestor of bilateral animals, the ARAS expansion did not occur until much later, in early vertebrate evolution. We propose that the surge in ARASs and consequent increase in AMPAR functionalities, contributed to the increased complexity of vertebrate brains and cognitive functions.
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http://dx.doi.org/10.1098/rsob.200234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653359PMC
October 2020

SynGAP splice variants display heterogeneous spatio-temporal expression and subcellular distribution in the developing mammalian brain.

J Neurochem 2020 09 10;154(6):618-634. Epub 2020 Mar 10.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain.

The SynGAP protein is a major regulator of synapse biology and neural circuit function. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic function and neural excitability. SynGAP has been involved in multiple signaling pathways and can regulate small GTPases with very different roles. Yet, the molecular bases behind this pleiotropy are poorly understood. We hypothesize that different SynGAP isoforms will mediate different sets of functions and that deciphering their spatio-temporal expression and subcellular localization will accelerate understanding their multiple functions. Using isoform-specific antibodies recognizing SynGAP in mouse and human samples we found distinctive developmental expression patterns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic density-95, in cortex and hippocampus during the first 2 weeks of postnatal development. Suggesting that during this period other isoforms would have a more prominent role. Furthermore, we observed subcellular localization differences between isoforms, particularly throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, SynGAP was predominantly found in non-synaptic locations in a period of early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms were always found enriched in the postsynaptic density, α2 isoforms changed from a non-synaptic to a mostly postsynaptic density localization with age and β isoforms were always found enriched in non-synaptic locations. The differential expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.
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http://dx.doi.org/10.1111/jnc.14988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754318PMC
September 2020

L-Serine dietary supplementation is associated with clinical improvement of loss-of-function -related pediatric encephalopathy.

Sci Signal 2019 06 18;12(586). Epub 2019 Jun 18.

Bellvitge Biomedical Research Institute (IDIBELL)-Unit of Neuropharmacology and Pain, University of Barcelona, Barcelona 08908, Spain.

Autosomal dominant mutations in are associated with severe encephalopathy, but little is known about the pathophysiological outcomes and any potential therapeutic interventions. Genetic studies have described the association between de novo mutations of genes encoding the subunits of the -methyl-d-aspartate receptor (NMDAR) and severe neurological conditions. Here, we evaluated a missense mutation in , causing a proline-to-threonine switch (P553T) in the GluN2B subunit of NMDAR, which was found in a 5-year-old patient with Rett-like syndrome with severe encephalopathy. Structural molecular modeling predicted a reduced pore size of the mutant GluN2B-containing NMDARs. Electrophysiological recordings in a HEK-293T cell line expressing the mutated subunit confirmed this prediction and showed an associated reduced glutamate affinity. Moreover, GluN2B(P553T)-expressing primary murine hippocampal neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of the AMPA receptor subunit GluA1 at stimulated synapses. Furthermore, the naturally occurring coagonist d-serine restored function to GluN2B(P553T)-containing NMDARs. l-Serine dietary supplementation of the patient was hence initiated, resulting in the increased abundance of d-serine in the plasma and brain. The patient has shown notable improvements in motor and cognitive performance and communication after 11 and 17 months of l-serine dietary supplementation. Our data suggest that l-serine supplementation might ameliorate -related severe encephalopathy and other neurological conditions caused by glutamatergic signaling deficiency.
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http://dx.doi.org/10.1126/scisignal.aaw0936DOI Listing
June 2019

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Neuron 2019 07 3;103(2):217-234.e4. Epub 2019 Jun 3.

Department of Functional Genomics, CNCR, VU University and UMC Amsterdam, 1081 HV Amsterdam, the Netherlands. Electronic address:

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).
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http://dx.doi.org/10.1016/j.neuron.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764089PMC
July 2019

Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid.

Mol Cell Proteomics 2019 Mar 22;18(3):546-560. Epub 2020 Sep 22.

§Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain;; ¶Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025Barcelona, Spain.

A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, p < 0.04), a finding that was replicated (0.7 to 0.8-fold, p < 0.05) for 6 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.
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http://dx.doi.org/10.1074/mcp.RA118.001290DOI Listing
March 2019

Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid.

Mol Cell Proteomics 2019 03 3;18(3):546-560. Epub 2019 Jan 3.

§Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain;

A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsynytenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages ( = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold ( < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, = 0.04) in an independent cohort ( = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, < 0.04), a finding that was replicated (0.7 to 0.8-fold, < 0.05) for 6 proteins in a third cohort ( = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold ( < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.
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http://dx.doi.org/10.1074/mcp.RA118.001290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398205PMC
March 2019

Synaptic metabolism and brain circuitries in inborn errors of metabolism.

J Inherit Metab Dis 2018 11;41(6):909-910

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain.

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http://dx.doi.org/10.1007/s10545-018-00252-yDOI Listing
November 2018

Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events.

Elife 2018 11 22;7. Epub 2018 Nov 22.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, Barcelona, Spain.

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings.
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http://dx.doi.org/10.7554/eLife.35774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307864PMC
November 2018

Characterization of the TLR Family in and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus.

Front Immunol 2018 2;9:2525. Epub 2018 Nov 2.

Department of Cell Biology, Animal Physiology and Immunology, Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra, Spain.

Toll-like receptors (TLRs) are important for raising innate immune responses in both invertebrates and vertebrates. Amphioxus belongs to an ancient chordate lineage which shares key features with vertebrates. The genomic research on TLR genes in and reveals the expansion of TLRs in amphioxus. However, the repertoire of TLRs in has not been studied and the functionality of amphioxus TLRs has not been reported. We have identified from transcriptomic data 30 new putative TLRs in and all of them are transcribed in adult amphioxus. Phylogenetic analysis showed that the repertoire of TLRs consists of both non-vertebrate and vertebrate-like TLRs. It also indicated a lineage-specific expansion in orthologous clusters of the vertebrate TLR11 family. We did not detect any representatives of the vertebrate TLR1, TLR3, TLR4, TLR5 and TLR7 families. To gain insight into these TLRs, we studied in depth a particular TLR highly similar to a gene annotated as bbtTLR1. The phylogenetic analysis of this novel BlTLR showed that it clusters with the vertebrate TLR11 family and it might be more related to TLR13 subfamily according to similar domain architecture. Transient and stable expression in HEK293 cells showed that the BlTLR localizes on the plasma membrane, but it did not respond to the most common mammalian TLR ligands. However, when the ectodomain of BlTLR is fused to the TIR domain of human TLR2, the chimeric protein could indeed induce NF-κB transactivation in response to the viral ligand Poly I:C, also indicating that in amphioxus, specific accessory proteins are needed for downstream activation. Based on the phylogenetic, subcellular localization and functional analysis, we propose that the novel BlTLR might be classified as an antiviral receptor sharing at least partly the functions performed by vertebrate TLR22. TLR22 is thought to be viral teleost-specific TLR but here we demonstrate that teleosts and amphioxus TLR22-like probably shared a common ancestor. Additional functional studies with other lancelet TLR genes will enrich our understanding of the immune response in amphioxus and will provide a unique perspective on the evolution of the immune system.
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http://dx.doi.org/10.3389/fimmu.2018.02525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224433PMC
October 2019

Setting the stage for a role of the postsynaptic proteome in inherited neurometabolic disorders.

Authors:
Àlex Bayés

J Inherit Metab Dis 2018 11 21;41(6):1093-1101. Epub 2018 Aug 21.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), C/Sant Antoni M. Claret, 167, 08025, Barcelona, Spain.

Neurotransmitter diseases are a well-defined group of metabolic conditions caused, in most instances, by genes specifically expressed in the presynaptic button. Better understanding of presynaptic molecular physiology, both in normal and pathological conditions, should help develop therapeutical strategies. The clinical relevance of the presynapse in inherited metabolic disorders is in glaring contrast with that of the postsynaptic component, which so far does not seem to play a relevant role in these disorders. This is somewhat surprising, as postsynaptic proteins are known to be involved in many nervous system diseases, particularly in neurodevelopmental and psychiatric disorders. The goal of this article is to explore if defects in the sophisticated postsynaptic machinery could also have a role in neurometabolic disorders.
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http://dx.doi.org/10.1007/s10545-018-0240-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326985PMC
November 2018

Chronic treatment with a MEK inhibitor reverses enhanced excitatory field potentials in Syngap1 mice.

Pharmacol Rep 2018 Aug 23;70(4):777-783. Epub 2018 Jun 23.

Centre for Clinical Brain Science, University of Edinburgh, Edinburgh, UK.

Background: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-d-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1 mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency.

Methods: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7-8-month old Syngap1 mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6days.

Results: We found that in hippocampal slices from vehicle-treated Syngap1 mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized basal synaptic responses, but did not reverse LTP deficit.

Conclusions: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals.
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http://dx.doi.org/10.1016/j.pharep.2018.02.021DOI Listing
August 2018

Synaptic proteomics as a means to identify the molecular basis of mental illness: Are we getting there?

Prog Neuropsychopharmacol Biol Psychiatry 2018 06 20;84(Pt B):353-361. Epub 2017 Sep 20.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Mª Claret 167, 08025 Barcelona, Spain; Universitat Autònoma de Barcelona, 08193, Bellaterra, Cerdanyola del Vallès, Spain\. Electronic address:

Synapses are centrally involved in many brain disorders, particularly in psychiatric and neurodevelopmental ones. However, our current understanding of the proteomic alterations affecting synaptic performance in the majority of mental illnesses is limited. As a result, novel pharmacotherapies with improved neurological efficacy have been scarce over the past decades. The main goal of synaptic proteomics in the context of mental illnesses is to identify dysregulated molecular mechanisms underlying these conditions. Here we reviewed and performed a meta-analysis of previous neuroproteomic research to identify proteins that may be consistently dysregulated in one or several mental disorders. Notably, we found very few proteins reproducibly altered among independent experiments for any given condition or between conditions, indicating that we are still far from identifying key pathophysiological mechanisms of mental illness. We suggest that future research in the field will require higher levels of standardization and larger-scale experiments to address the challenge posed by biological and methodological variability. We strongly believe that more resources should be placed in this field as the need to identify the molecular roots of mental illnesses is highly pressing.
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http://dx.doi.org/10.1016/j.pnpbp.2017.09.011DOI Listing
June 2018

Rett-like Severe Encephalopathy Caused by a De Novo GRIN2B Mutation Is Attenuated by D-serine Dietary Supplement.

Biol Psychiatry 2018 Jan 16;83(2):160-172. Epub 2017 Jun 16.

Bellvitge Biomedical Research Institute-Unit of Neuropharmacology and Pain Group, University of Barcelona, Barcelona, Spain. Electronic address:

Background: N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR.

Methods: We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice.

Results: Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17 months of D-serine dietary supplementation.

Conclusions: Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.
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http://dx.doi.org/10.1016/j.biopsych.2017.05.028DOI Listing
January 2018

Evolution of complexity in the zebrafish synapse proteome.

Nat Commun 2017 03 2;8:14613. Epub 2017 Mar 2.

Genes to Cognition Programme, Centre for Clinical Brain Science, University of Edinburgh, Edinburgh EH16 4SB, UK.

The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.
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http://dx.doi.org/10.1038/ncomms14613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337974PMC
March 2017

Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes.

Mol Brain 2014 Nov 28;7:88. Epub 2014 Nov 28.

Background: Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown.

Results: Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases.

Conclusions: We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.
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http://dx.doi.org/10.1186/s13041-014-0088-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271336PMC
November 2014

Glutamate receptor mutations in psychiatric and neurodevelopmental disorders.

Commun Integr Biol 2014 Jan 6;7(1):e27887. Epub 2014 Feb 6.

Molecular Physiology of the Synapse Laboratory; Biomedical Research Institute Sant Pau (IIB Sant Pau); Barcelona, Spain ; Universitat Autònoma de Barcelona; Bellaterra (Cerdanyola del Vallès), Spain.

Alterations in glutamatergic neurotransmission have long been associated with psychiatric and neurodevelopmental disorders (PNDD), but only recent advances in high-throughput DNA sequencing have allowed interrogation of the prevalence of mutations in glutamate receptors (GluR) among afflicted individuals. In this review we discuss recent work describing GluR mutations in the context of PNDDs. Although there are no strict relationships between receptor subunit or type and disease, some interesting preliminary conclusions have arisen. Mutations in genes coding for ionotropic glutamate receptor subunits, which are central to synaptic transmission and plasticity, are mostly associated with intellectual disability and autism spectrum disorders. In contrast, mutations of metabotropic GluRs, having a role on modulating neural transmission, are preferentially associated with psychiatric disorders. Also, the prevalence of mutations among GluRs is highly heterogeneous, suggesting a critical role of certain subunits in PNDD pathophysiology. The emerging bias between GluR subtypes and specific PNDDs may have clinical implications.
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http://dx.doi.org/10.4161/cib.27887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937208PMC
January 2014

Zinc transporter-1 concentrates at the postsynaptic density of hippocampal synapses.

Mol Brain 2014 Mar 7;7:16. Epub 2014 Mar 7.

Department of Pharmacology, University of Barcelona, Barcelona 08036, Spain.

Background: Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses.

Findings: Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons.

Conclusions: Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.
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http://dx.doi.org/10.1186/1756-6606-7-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975337PMC
March 2014

Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins.

PLoS One 2012 5;7(10):e46683. Epub 2012 Oct 5.

Molecular Physiology of the Synapse Laboratory, Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, UAB, Barcelona, Catalonia, Spain.

Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046683PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465276PMC
April 2013

Characterization of the proteome, diseases and evolution of the human postsynaptic density.

Nat Neurosci 2011 Jan 19;14(1):19-21. Epub 2010 Dec 19.

Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, UK.

We isolated the postsynaptic density from human neocortex (hPSD) and identified 1,461 proteins. hPSD mutations cause 133 neurological and psychiatric diseases and were enriched in cognitive, affective and motor phenotypes underpinned by sets of genes. Strong protein sequence conservation in mammalian lineages, particularly in hub proteins, indicates conserved function and organization in primate and rodent models. The hPSD is an important structure for nervous system disease and behavior.
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http://dx.doi.org/10.1038/nn.2719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040565PMC
January 2011

In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.

J Neurosci 2010 Jun;30(24):8162-70

Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom.

Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.
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http://dx.doi.org/10.1523/JNEUROSCI.1792-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912510PMC
June 2010

Neuroproteomics: understanding the molecular organization and complexity of the brain.

Nat Rev Neurosci 2009 Sep;10(9):635-46

Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

Advances in technology have equipped the field of neuroproteomics with refined tools for the study of the expression, interaction and function of proteins in the nervous system. In combination with bioinformatics, neuroproteomics can address the organization of dynamic, functional protein networks and macromolecular structures that underlie physiological, anatomical and behavioural processes. Furthermore, neuroproteomics is contributing to the elucidation of disease mechanisms and is a powerful tool for the identification of biomarkers.
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http://dx.doi.org/10.1038/nrn2701DOI Listing
September 2009

Evolutionary expansion and anatomical specialization of synapse proteome complexity.

Nat Neurosci 2008 Jul 8;11(7):799-806. Epub 2008 Jun 8.

Institute for Science and Technology in Medicine, Keele University, Thornburrow Drive, Hartshill, Stoke-on-Trent ST4 7QB, UK.

Understanding the origins and evolution of synapses may provide insight into species diversity and the organization of the brain. Using comparative proteomics and genomics, we examined the evolution of the postsynaptic density (PSD) and membrane-associated guanylate kinase (MAGUK)-associated signaling complexes (MASCs) that underlie learning and memory. PSD and MASC orthologs found in yeast carry out basic cellular functions to regulate protein synthesis and structural plasticity. We observed marked changes in signaling complexity at the yeast-metazoan and invertebrate-vertebrate boundaries, with an expansion of key synaptic components, notably receptors, adhesion/cytoskeletal proteins and scaffold proteins. A proteomic comparison of Drosophila and mouse MASCs revealed species-specific adaptation with greater signaling complexity in mouse. Although synaptic components were conserved amongst diverse vertebrate species, mapping mRNA and protein expression in the mouse brain showed that vertebrate-specific components preferentially contributed to differences between brain regions. We propose that the evolution of synapse complexity around a core proto-synapse has contributed to invertebrate-vertebrate differences and to brain specialization.
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http://dx.doi.org/10.1038/nn.2135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624047PMC
July 2008

Caught after the Act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.

Biochemistry 2007 Jun 17;46(23):6921-30. Epub 2007 May 17.

Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Spain.

A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1', influence Km and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 A resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S5-S1' and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights.
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http://dx.doi.org/10.1021/bi700480bDOI Listing
June 2007

Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.

Insect Biochem Mol Biol 2006 Aug 26;36(8):654-64. Epub 2006 May 26.

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.
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http://dx.doi.org/10.1016/j.ibmb.2006.05.010DOI Listing
August 2006

Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors.

Proc Natl Acad Sci U S A 2005 Nov 31;102(46):16602-7. Epub 2005 Oct 31.

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.

Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase alpha/beta-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1' substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the beta8-alpha9 loop, and alpha7 together with the alpha7-alpha8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.
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http://dx.doi.org/10.1073/pnas.0505489102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1283804PMC
November 2005

Primary purulent pericarditis due to group C Streptococcus.

Can J Cardiol 2004 Dec;20(14):1479-80

CSTAR-University Hospital, 339 Windermere Road, London, Ontario N6A 5A5, Canada.

In the antibiotic era, purulent pericarditis, an infection associated with high mortality, is uncommon. The causative organism is generally Staphylococcus aureus or Streptococcus pneumoniae arising from contiguous spread or hematogenous dissemination of an underlying infection elsewhere in the body. The present report describes a previously healthy individual who presented with acute infectious pericarditis with the offending organism identified as Lancefield group C Streptococcus equi. After an initial pericardial window was unable to prevent recurrent pericardial effusion, pericardiectomy was performed and the patient slowly recovered from the incident.
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December 2004

Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation.

Biol Chem 2004 Jun;385(6):517-24

Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS. The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244). Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6. A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production. Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism. Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.
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http://dx.doi.org/10.1515/BC.2004.061DOI Listing
June 2004

Procarboxypeptidase A from the insect pest Helicoverpa armigera and its derived enzyme. Two forms with new functional properties.

Eur J Biochem 2003 Jul;270(14):3026-35

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, Spain.

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a Ki in the pm range.
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http://dx.doi.org/10.1046/j.1432-1033.2003.03681.xDOI Listing
July 2003

The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family.

J Biol Chem 2002 Jul 16;277(30):27273-81. Epub 2002 May 16.

Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Cientificas, c/Jordi Girona 18-26, Barcelona 08034, Spain.

Zyme/protease M/neurosin/human kallikrein 6 (hK6) is a member of the human kallikrein family of trypsin-like serine proteinases and was originally identified as being down-regulated in metastatic breast and ovarian tumors when compared with corresponding primary tumors. Recent evidence suggests that hK6 may serve as a circulating tumor marker in ovarian cancers. In addition, it was described in the brain of Parkinson's disease and Alzheimer's disease patients, where it is implicated in amyloid precursor protein processing. It is thus a biomarker for these diseases. To examine the mechanism of activation of hK6, we have solved the structure of its proform, the first of a human kallikrein family member. The proenzyme displays a fold that exhibits chimeric features between those of trypsinogen and other family members. It lacks the characteristic "kallikrein loop" and forms the six disulfide bridges of trypsin. Pro-hK6 displays a completely closed specificity pocket and a unique conformation of the regions involved in structural rearrangements upon proteolytic cleavage activation. This points to a novel activation mechanism, which could be extrapolated to other human kallikreins.
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http://dx.doi.org/10.1074/jbc.M201534200DOI Listing
July 2002