ABSTRACT
Cardiometabolic diseases, such as type 2 diabetes and cardiovascular disease, have a high public health burden. Understanding the genetically determined regulation of proteins that are dysregulated in disease can help to dissect the complex biology underpinning them. Here, we perform a protein quantitative trait locus (pQTL) analysis of 248 serum proteins relevant to cardiometabolic processes in 2893 individuals. Meta-analyzing whole-genome sequencing (WGS) data from two Greek cohorts, MANOLIS (n = 1356; 22.5× WGS) and Pomak (n = 1537; 18.4× WGS), we detect 301 independently associated pQTL variants for 170 proteins, including 12 rare variants (minor allele frequency < 1%). We additionally find 15 pQTL variants that are rare in non-Finnish European populations but have drifted up in the frequency in the discovery cohorts here. We identify proteins causally associated with cardiometabolic traits, including Mep1b for high-density lipoprotein (HDL) levels, and describe a knock-out (KO) Mep1b mouse model. Our findings furnish insights into the genetic architecture of the serum proteome, identify new protein-disease relationships and demonstrate the importance of isolated populations in pQTL analysis.
Subject(s)
Cardiovascular Diseases , Diabetes Mellitus, Type 2 , Animals , Mice , Phenotype , Whole Genome Sequencing , Blood Proteins/genetics , Genome-Wide Association StudyABSTRACT
Neurotoxic amyloid-ß (Aß) peptides cause neurodegeneration in Alzheimer's disease (AD) patients' brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the ß-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aß species in the mouse brains differs from those observed in AD patients' brains. Particularly mutations proximal to the ß-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aß1-x formation) lead to artificial Aß production, as N-terminally truncated Aß peptides are hardly present in these mouse brains. Meprin ß is an alternative ß-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aß2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aß2-x peptides by conditionally overexpressing meprin ß in astrocytes. We chose astrocytes as meprin ß was detected in this cell type in close proximity to Aß plaques in AD patients' brains. The meprin ß-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aß production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aß species in future studies.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Humans , Mice , Animals , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Astrocytes/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Proteolysis , Brain/metabolismABSTRACT
Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-ß peptides (Aß42 and/or Aß43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aß production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aß42/Aß40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aß42/Aß40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aß production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid Precursor Protein Secretases/metabolism , Mutant Proteins/genetics , Mutation , Peptide Fragments/metabolism , Presenilin-1/metabolism , Animals , MiceABSTRACT
The acute ischemic stroke therapy of choice is the application of Alteplase, a drug containing the enzyme tissue-type plasminogen activator (tPa) which rapidly destabilizes blood clots. A central hallmark of stroke pathology is blood-brain barrier (BBB) breakdown associated with tight junction (TJ) protein degradation, which seems to be significantly more severe under therapeutic conditions. The exact mechanisms how tPa facilitates BBB breakdown are not entirely understood. There is evidence that an interaction with the lipoprotein receptor-related protein 1 (LRP1), allowing tPa transport across the BBB into the central nervous system, is necessary for this therapeutic side effect. Whether tPa-mediated disruption of BBB integrity is initiated directly on microvascular endothelial cells or other brain cell types is still elusive. In this study we could not observe any changes of barrier properties in microvascular endothelial cells after tPa incubation. However, we present evidence that tPa causes changes in microglial activation and BBB breakdown after LRP1-mediated transport across the BBB. Using a monoclonal antibody targeting the tPa binding sites of LRP1 decreased tPa transport across an endothelial barrier. Our results indicate that limiting tPa transport from the vascular system into the brain by coapplication of a LRP1-blocking monoclonal antibody might be a novel approach to minimize tPa-related BBB damage during acute stroke therapy.
Subject(s)
Ischemic Stroke , Stroke , Humans , Tissue Plasminogen Activator/adverse effects , Tissue Plasminogen Activator/metabolism , Endothelial Cells/metabolism , Ischemic Stroke/chemically induced , Ischemic Stroke/complications , Ischemic Stroke/drug therapy , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Low Density Lipoprotein Receptor-Related Protein-1/therapeutic use , Stroke/drug therapy , Stroke/pathology , Antibodies, Monoclonal/therapeutic use , Lipoproteins, LDLABSTRACT
Despite the neurodegenerative disorder Alzheimer's disease (AD) is the most common form of dementia in late adult life, there is currently no therapy available to prevent the onset or slow down the progression of AD. The progressive cognitive decline in AD correlates with a successive accumulation of cerebral amyloid-ß (Aß) due to impaired clearance mechanisms. A significant percentage is removed by low-density lipoprotein receptor-related protein 1 (LRP1)-mediated transport across the blood-brain barrier (BBB) into the periphery. Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to members of the low-density lipoprotein receptor protein family at the cell surface and targets them for lysosomal degradation, which reduces the number of functional receptors. However, the adverse impact of PCSK9 on LRP1-mediated brain Aß clearance remains elusive. By using an established BBB model, we identified reduced LRP1-mediated brain-to-blood Aß clearance due to PCSK9 across different endothelial monolayer in vitro. Consequently, the repetitive application of FDA-approved monoclonal anti-PCSK9 antibodies into 5xFAD mice decreased the cerebral Aß burden across variants and aggregation state, which was not reproducible in brain endothelial-specific LRP1-/- 5xFAD mice. The peripheral PCSK9 inhibition reduced Aß pathology in prefrontal cortex and hippocampus-brain areas critically involved in memory processing-and prevented disease-related impairment in hippocampus-dependent memory formation. Our data suggest that peripheral inhibition of PCSK9 by already available therapeutic antibodies may be a novel and easily applicable potential AD treatment.
Subject(s)
Blood-Brain Barrier , Proprotein Convertase 9 , Amyloid beta-Peptides/metabolism , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Humans , Mice , Proprotein Convertase 9/metabolismABSTRACT
ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Brain/metabolism , Learning , Memory Disorders/pathology , Metalloendopeptidases/deficiency , Aged , Amyloid Precursor Protein Secretases/metabolism , Animals , Astrocytes/metabolism , Brain/pathology , Crosses, Genetic , Disease Models, Animal , Female , Glial Fibrillary Acidic Protein/metabolism , Humans , Male , Metalloendopeptidases/metabolism , Mice, Knockout , Peptides/metabolism , Protein Processing, Post-TranslationalABSTRACT
Meprin ß is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin ß including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin ß is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin ß including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin ß with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin ß from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin ß was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin ß phosphorylation and identified the involvement of PKC-α and PKC-ß. As a result of phosphorylation, the meprin ß activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin ß.
Subject(s)
Cell Membrane/metabolism , Colonic Neoplasms/pathology , Extracellular Space/metabolism , Metalloendopeptidases/metabolism , Protein Kinase C beta/metabolism , Protein Kinase C-alpha/metabolism , Proteolysis , Colonic Neoplasms/metabolism , Gene Expression Regulation , Humans , Metalloendopeptidases/genetics , Phosphorylation , Protein Kinase C beta/genetics , Protein Kinase C-alpha/genetics , Tumor Cells, CulturedABSTRACT
The accumulation of neurotoxic amyloid-beta (Aß) in the brain is one of the characteristic hallmarks of Alzheimer's disease (AD). Aß-peptide brain homeostasis is governed by its production and various clearance mechanisms. The blood-brain barrier provides a large surface area for influx and efflux mechanisms into and out of the brain. Different transporters and receptors have been implicated to play crucial roles in Aß clearance from brain. Besides Aß transport, the blood-brain barrier tightly regulates the brain's microenvironment; however, vascular alterations have been shown in patients with AD. Here, we summarize how the blood-brain barrier changes during aging and in disease and focus on recent findings of how the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) play a role in Aß clearance from brain.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Blood-Brain Barrier/metabolism , Brain/metabolism , Humans , Peptide Fragments/metabolism , Receptors, LDL/metabolismABSTRACT
Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.
Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Membrane Proteins/metabolism , Metalloproteases/metabolism , Amino Acids/metabolism , Animals , Cell Line , HEK293 Cells , Humans , Mice , Myocytes, Cardiac/metabolismABSTRACT
The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.
Subject(s)
Blood-Brain Barrier/metabolism , Leptin/metabolism , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Obesity/metabolism , Obesity/pathology , Animals , Binding Sites , Body Composition , Body Weight , CHO Cells , Choroid Plexus/metabolism , Cricetulus , Endothelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Luciferases/metabolism , Male , Models, Biological , Phosphorylation , Protein Transport , Receptors, Leptin/metabolism , Recombinant Fusion Proteins/metabolism , SwineABSTRACT
The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.
Subject(s)
Ependymoglial Cells , Animals , Astrocytes , Female , Lipoproteins, LDL , Male , Mice , Prosencephalon , Receptors, Lipoprotein , Seizures , Tissue Plasminogen ActivatorABSTRACT
Biologic activity of proteases is mainly characterized by the substrate specificity, tissue distribution, and cellular localization. The human metalloproteases meprin α and meprin ß share 41% sequence identity and exhibit a similar cleavage specificity with a preference for negatively charged amino acids. However, shedding of meprin α by furin on the secretory pathway makes it a secreted enzyme in comparison with the membrane-bound meprin ß. In this study, we identified human meprin α and meprin ß as forming covalently linked membrane-tethered heterodimers in the early endoplasmic reticulum, thereby preventing furin-mediated secretion of meprin α. Within this newly formed enzyme complex, meprin α was able to be activated on the cell surface and detected by cleavage of a novel specific fluorogenic peptide substrate. However, the known meprin ß substrates amyloid precursor protein and CD99 were not shed by membrane-tethered meprin α. On the other hand, being linked to meprin α, activation of or substrate cleavage by meprin ß on the cell surface was not altered. Interestingly, proteolytic activity of both proteases was increased in the heteromeric complex, indicating an increased proteolytic potential at the plasma membrane. Because meprins are susceptibility genes for inflammatory bowel disease (IBD), and to investigate the physiologic impact of the enzyme complex, we performed transcriptome analyses of intestinal mucosa from meprin-knockout mice. Comparison of the transcriptional gene analysis data with gene analyses of IBD patients revealed that different gene subsets were dysregulated if meprin α was expressed alone or in the enzyme complex, demonstrating the physiologic and pathophysiological relevance of the meprin heterodimer formation.-Peters, F., Scharfenberg, F., Colmorgen, C., Armbrust, F., Wichert, R., Arnold, P., Potempa, B., Potempa, J., Pietrzik, C. U., Häsler, R., Rosenstiel, P., Becker-Pauly, C. Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes.
Subject(s)
Inflammatory Bowel Diseases/genetics , Metalloendopeptidases/metabolism , Animals , Cell Membrane/metabolism , HeLa Cells , Humans , Metalloendopeptidases/genetics , Mice , Mice, KnockoutABSTRACT
Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-ß (Aß) plaques are one of the pathological hallmarks in AD patient brains. Aß accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. However, so far disease-modifying drugs targeting ß- and γ-secretase pathways seeking a decrease in the production of toxic Aß peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin ß acts as an alternative ß-secretase, capable of generating truncated Aß2-x peptides that have been described to be increased in AD patients. This indicates an important ß-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin ß within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin ß itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin ß. Overall, we highlight the current cellular, molecular and structural understanding of meprin ß as alternative ß-secretase within the complex protease web, regulating APP processing in health and disease.
Subject(s)
ADAM10 Protein/metabolism , Metalloendopeptidases/metabolism , ADAM10 Protein/chemistry , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Humans , Membrane Proteins/metabolism , Metalloendopeptidases/chemistry , Presenilin-1/metabolism , Proteolysis , Serine Endopeptidases/metabolismABSTRACT
Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.
Subject(s)
ADAMTS4 Protein/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Oligodendroglia/metabolism , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/metabolism , Animals , Brain/metabolism , Brain/pathology , Disease Models, Animal , HEK293 Cells , Humans , Mice , Oligodendroglia/pathology , Peptide Fragments/metabolism , Plaque, Amyloid/pathologyABSTRACT
Amyloid-ß peptides (Aß) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aß metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aß. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aß metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aß oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aß clearance across the pBCEC monolayer. Treatment of pBCEC with Aß(1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aß oligomers and reduced Aß uptake and cell-associated Aß oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aß processing and clearance at the BBB.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Blood-Brain Barrier/drug effects , Clusterin/pharmacology , Endothelial Cells/drug effects , Protein Processing, Post-Translational/drug effects , Simvastatin/pharmacology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/chemistry , Animals , Blood-Brain Barrier/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/metabolism , SwineABSTRACT
The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin ß and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin ß with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin ß would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin ß induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin ß in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin ß could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin ß inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.
Subject(s)
12E7 Antigen/metabolism , Conserved Sequence , Metalloendopeptidases/metabolism , Proteolysis , Transendothelial and Transepithelial Migration , 12E7 Antigen/chemistry , Animals , Carcinoma, Lewis Lung/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BLABSTRACT
The accumulation of neurotoxic amyloid-beta (Aß) in the brain is a characteristic hallmark of Alzheimer's disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aß. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aß efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aß through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aß transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aß from the brain.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Monomeric Clathrin Assembly Proteins/physiology , Receptors, LDL/metabolism , Tumor Suppressor Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/physiology , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/physiology , Brain/metabolism , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/physiology , Low Density Lipoprotein Receptor-Related Protein-1 , Male , Mice , Mice, Knockout , Monomeric Clathrin Assembly Proteins/metabolism , Peptide Fragments/metabolism , Primary Cell Culture , Receptors, LDL/physiology , Swine , Transcytosis/physiology , Tumor Suppressor Proteins/physiologyABSTRACT
Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.
Subject(s)
Benzoxazines/chemistry , Serine Proteinase Inhibitors/chemistry , Styrenes/chemistry , Animals , Benzoxazines/chemical synthesis , Catalytic Domain , Cattle , Chymotrypsin/chemistry , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Drosophila/chemistry , Drosophila Proteins/metabolism , Drug Discovery , Endopeptidases/chemistry , Endopeptidases/genetics , Enzyme Assays , Escherichia coli/enzymology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Docking Simulation , Mutation , Serine/chemistry , Serine Proteinase Inhibitors/chemical synthesis , Styrenes/chemical synthesis , Transforming Growth Factor alpha/metabolismABSTRACT
Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.
Subject(s)
Drug Design , Saccharin/analogs & derivatives , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Computer-Aided Design , HEK293 Cells , Humans , Membrane Proteins , Saccharin/pharmacology , Serine Proteinase Inhibitors/chemistryABSTRACT
The accumulation of the neurotoxin beta-amyloid (Aß) is a major hallmark in Alzheimer's disease (AD). Aß homeostasis in the brain is governed by its production and various clearance mechanisms. Both pathways are influenced by the ubiquitously expressed low-density lipoprotein receptor-related protein 1 (LRP1). In cerebral blood vessels, LRP1 is an important mediator for the rapid removal of Aß from brain via transport across the blood-brain barrier (BBB). Here, we summarize recent findings on LRP1 function and discuss the targeting of LRP1 as a modulator for AD pathology and drug delivery into the brain.