ABSTRACT
The accumulation of ß-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha-synuclein and ß-amyloid fibrillation in vitro and is up-regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.
ABSTRACT
AIMS/HYPOTHESIS: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. METHODS: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (ßS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. RESULTS: ßS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in ßS2KO islets. Islets isolated from ßS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of ßS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in ßS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. CONCLUSIONS/INTERPRETATION: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. DATA AVAILABILITY: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).
Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Islets of Langerhans , Mice , Humans , Animals , Proinsulin/genetics , Proinsulin/metabolism , Insulin-Secreting Cells/metabolism , Diabetes Mellitus, Type 2/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Insulin/metabolism , Glucose/metabolism , Islets of Langerhans/metabolismABSTRACT
Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.
Subject(s)
Furin , Proprotein Convertases , Amino Acid Sequence , Carbamates , Fluorescent Dyes , Oligopeptides , Proteins , Subtilisins/metabolismABSTRACT
BACKGROUND: During the last two decades, over 100 proteomics studies have identified a variety of potential biomarkers in CSF of Alzheimer's (AD) patients. Although several reviews have proposed specific biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specific up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteomics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identified in each study are consistent in distribution; direction change; and significance, which would strengthen their potential use in studies of AD pathology and progression. METHODS: We generated a database containing all CSF proteins whose levels are known to be significantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. RESULTS: Of the 162 proteins that were identified in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identified 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fingerprinting revealed specific peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. CONCLUSIONS: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical significance; this panel constitutes a potent tool for the classification and diagnosis of AD.
ABSTRACT
Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aß peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aß failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.
Subject(s)
PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/pathology , Prions/metabolism , alpha-Synuclein/metabolism , Animals , Cricetinae , Humans , Mesocricetus , Mice , Protein FoldingABSTRACT
Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson's disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson's disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158-180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral dopaminergic neurons, and recombinant proSAAS blocks α-synuclein-induced cytotoxicity in SH-SY5Y cells. Four independent proteomics studies have previously identified proSAAS as a potential cerebrospinal fluid biomarker in various neurodegenerative diseases. Coupled with prior work showing that proSAAS blocks ß-amyloid aggregation into fibrils, this study supports the idea that neuronal proSAAS plays an important role in proteostatic processes. ProSAAS thus represents a possible therapeutic target in neurodegenerative disease.
Subject(s)
Neuropeptides/physiology , alpha-Synuclein/metabolism , Animals , Cells, Cultured , Humans , Lewy Bodies/metabolism , Neuropeptides/chemistry , Neurotoxicity Syndromes/prevention & control , Protein Aggregates , Protein Multimerization , Rats , Substantia Nigra/metabolism , alpha-Synuclein/toxicityABSTRACT
Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of ß-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying ß-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in ß cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.
Subject(s)
Carboxypeptidase H/metabolism , Diabetes Mellitus, Type 2/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Insulin-Secreting Cells/physiology , Insulin/metabolism , Animals , Carboxypeptidase H/genetics , Cells, Cultured , Diabetes Mellitus, Type 2/genetics , Endoplasmic Reticulum Stress/physiology , Eukaryotic Initiation Factor-4G/genetics , Genome-Wide Association Study , Homeodomain Proteins/metabolism , Humans , Insulin-Secreting Cells/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Biosynthesis/physiology , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Signal Transduction/physiology , Sterol Regulatory Element Binding Protein 1/metabolism , Trans-Activators/metabolismABSTRACT
Cellular protein homeostasis (proteostasis) maintains the integrity of the proteome and includes protein synthesis, folding, oligomerization, and turnover; chaperone proteins assist with all of these processes. Neurons appear to be especially susceptible to failures in proteostasis, and this is now increasingly recognized as a major origin of neurodegenerative disease. This review, based on a mini-symposium presented at the 2015 Society for Neuroscience meeting, describes new work in the area of neuronal proteostasis, with a specific focus on the roles and therapeutic uses of protein chaperones. We first present a brief review of protein misfolding and aggregation in neurodegenerative disease. We then discuss different aspects of chaperone control of neuronal proteostasis on topics ranging from chaperone engineering, to chaperone-mediated blockade of protein oligomerization and cytotoxicity, to the potential rescue of neurodegenerative processes using modified chaperone proteins. SIGNIFICANCE STATEMENT: Aberrant protein homeostasis within neurons results in protein misfolding and aggregation. In this review, we discuss specific roles for protein chaperones in the oligomerization, assembly, and disaggregation of proteins known to be abnormally folded in neurodegenerative disease. Collectively, our goal is to identify therapeutic mechanisms to reduce the cellular toxicity of abnormal aggregates.
Subject(s)
Molecular Chaperones/metabolism , Neurodegenerative Diseases/metabolism , Animals , Humans , Molecular Chaperones/genetics , Neurodegenerative Diseases/geneticsABSTRACT
Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded ß-sheet-rich proteins, including amyloid-ß (Aß)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aß(1-42), Aß(1-40), and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aß(1-42) and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aß(1-42)-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aß plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Neurodegenerative Diseases/metabolism , Neuroendocrine Secretory Protein 7B2/physiology , alpha-Synuclein/antagonists & inhibitors , Aged , Amino Acid Sequence , Amyloid beta-Peptides/metabolism , Animals , Cell Death/physiology , Female , Hippocampus/metabolism , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Microscopy, Electron, Transmission , Molecular Sequence Data , Neuroendocrine Secretory Protein 7B2/chemistry , Neuroendocrine Secretory Protein 7B2/metabolism , Substantia Nigra/metabolism , alpha-Synuclein/metabolismABSTRACT
Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by an abnormal aggregation of misfolded beta-sheet rich proteins such as ß-amyloid (Aß). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti-aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12-month-old APdE9 mice, and in the cortex of a human AD-affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co-immunoprecipitated with Aß immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aß(1-42) at molar ratios of 1 : 10, and this anti-aggregation effect was dose dependent. Structure-function studies showed that residues 97-180 were sufficient for the anti-aggregation function against Aß. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral-mediated proSAAS over-expression, blocked the neurocytotoxic effect of Aß(1-42) in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimer's disease pathology.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid/drug effects , Nerve Tissue Proteins/physiology , Aged , Amyloid/chemistry , Amyloid/genetics , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/toxicity , Animals , Blotting, Western , Cell Line , Cell Survival/drug effects , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Lentivirus/genetics , Male , Mice , Mice, Transgenic , Microscopy, Electron , Molecular Chaperones , Nerve Tissue Proteins/genetics , Neuropeptides , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/toxicity , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Tissue FixationABSTRACT
BACKGROUND & AIMS: Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. METHODS: We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. RESULTS: We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. CONCLUSIONS: In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.
Subject(s)
Diarrhea/etiology , Endocrine System Diseases/etiology , Malabsorption Syndromes/etiology , Obesity/complications , Proprotein Convertase 1/deficiency , Adolescent , Adrenocorticotropic Hormone/blood , Child , Child, Preschool , Cohort Studies , Endocrine System Diseases/complications , Endocrine System Diseases/congenital , Female , Humans , Infant , Male , Mutation , Obesity/congenital , Proprotein Convertase 1/geneticsABSTRACT
The accumulation of ß-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha synuclein and ß-amyloid fibrillation in vitro, and is upregulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using Western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control (and/or reduced degradation). ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Interestingly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.
ABSTRACT
Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.
Subject(s)
Communicable Diseases/drug therapy , Furin/antagonists & inhibitors , Proprotein Convertases/antagonists & inhibitors , Animals , Benzamidines/chemistry , Chemistry, Pharmaceutical/methods , Chromatography, High Pressure Liquid/methods , Dogs , Dose-Response Relationship, Drug , Drug Design , Furin/chemistry , Hemagglutinins/chemistry , Humans , Kinetics , Micelles , Models, Chemical , Oligopeptides/chemistry , Peptide Hydrolases/chemistry , Peptides/chemistry , Protease Inhibitors/pharmacology , Saccharomyces cerevisiae/metabolism , Shiga Toxin/chemistryABSTRACT
Computational methods have led two groups to predict the endogenous presence of a highly conserved, amidated, 14-aa neuropeptide called either spexin or NPQ. NPQ/spexin is part of a larger prohormone that contains 3 sets of RR residues, suggesting that it could yield more than one bioactive peptide; however, no in vivo activity has been demonstrated for any peptide processed from this precursor. Here we demonstrate biological activity for two peptides present within proNPQ/spexin. NPQ/spexin (NWTPQAMLYLKGAQ-NH(2)) and NPQ 53-70 (FISDQSRRKDLSDRPLPE) have differing renal and cardiovascular effects when administered intracerebroventricularly or intravenously into rats. Intracerebroventricular injection of NPQ/spexin produced a 13 ± 2 mmHg increase in mean arterial pressure, a 38 ± 8 bpm decrease in heart rate, and a profound decrease in urine flow rate. Intracerebroventricular administration of NPQ 53-70 produced a 26 ± 9 bpm decrease in heart rate with no change in mean arterial pressure, and a marked increase in urine flow rate. Intraventricular NPQ/spexin and NPQ 53-70 also produced antinociceptive activity in the warm water tail withdrawal assay in mice (ED(50)<30 and 10 nmol for NPQ/spexin and NPQ 53-70, respectively). We conclude that newly identified peptides derived from the NPQ/spexin precursor contribute to CNS-mediated control of arterial blood pressure and salt and water balance and modulate nociceptive responses.
Subject(s)
Cardiovascular Physiological Phenomena , Kidney/physiology , Neuropeptides/physiology , Nociception/physiology , Peptide Hormones/physiology , Amino Acid Sequence , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Cardiovascular Physiological Phenomena/drug effects , Humans , Injections, Intraventricular , Kidney/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Molecular Sequence Data , Neuropeptides/administration & dosage , Neuropeptides/genetics , Nociception/drug effects , Peptide Fragments/administration & dosage , Peptide Fragments/genetics , Peptide Fragments/physiology , Peptide Hormones/administration & dosage , Peptide Hormones/genetics , Protein Processing, Post-Translational , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
OBJECTIVES: Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. METHODS: We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. RESULTS: Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. CONCLUSIONS: Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.
Subject(s)
Codon, Nonsense , Diabetes Insipidus/complications , Diarrhea/genetics , Exome , Malabsorption Syndromes/genetics , Proprotein Convertase 1/genetics , Diarrhea/congenital , Diarrhea/diagnosis , Homozygote , Humans , Infant, Newborn , Malabsorption Syndromes/congenital , Malabsorption Syndromes/diagnosis , Male , Proprotein Convertase 1/metabolism , Sequence Analysis, DNAABSTRACT
The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.
Subject(s)
Furin/antagonists & inhibitors , Single-Domain Antibodies/pharmacology , Animals , Antibody Specificity , Camelus , Catalysis/drug effects , Coumarins/metabolism , Diphtheria Toxin/metabolism , Endocytosis , Furin/chemistry , Furin/immunology , Furin/metabolism , Glypicans/metabolism , HEK293 Cells/metabolism , Humans , Kinetics , Mice , Oligopeptides/metabolism , Peptide Fragments/metabolism , Proprotein Convertases/metabolism , Protein Binding/drug effects , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Substrate Specificity , Transforming Growth Factor beta/metabolismABSTRACT
The small neuroendocrine protein 7B2 has been shown to be required for the productive maturation of proprotein convertase 2 (proPC2) to an active enzyme form; this action is accomplished via its ability to block aggregation of proPC2 into nonactivatable forms. Recent data show that 7B2 can also act as a postfolding chaperone to block the aggregation of a number of other proteins, for example, α-synuclein. To gain insight into the mechanism of action of 7B2 in blocking protein aggregation, we performed structural studies of this protein using gel filtration chromatography, intrinsic tryptophan fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Gel filtration studies indicated that 7B2 exists as an extended monomer, eluting at a molecular mass higher than that expected for a globular protein of similar size. However, chemical cross-linking showed that 7B2 exhibits concentration-dependent oligomerization. CD experiments showed that both full-length 27 kDa 7B2 and the C-terminally truncated 21 kDa form lack appreciable secondary structure, although the longer protein exhibited more structural content than the latter, as demonstrated by intrinsic and ANS fluorescence studies. NMR spectra confirmed the lack of structure in native 7B2, but a disorder-to-order transition was observed upon incubation with one of its client proteins, α-synuclein. We conclude that 7B2 is a natively disordered protein whose function as an antiaggregant chaperone is likely facilitated by its lack of appreciable secondary structure and tendency to form oligomers.
Subject(s)
Neuroendocrine Secretory Protein 7B2/chemistry , Chromatography, Gel , Circular Dichroism , Humans , Neuroendocrine Secretory Protein 7B2/isolation & purification , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.
Subject(s)
Enzyme Inhibitors/pharmacology , Hexosamines/pharmacology , Proprotein Convertases/antagonists & inhibitors , Animals , Cell Line , Inhibitory Concentration 50 , Kinetics , Mice , Proprotein Convertases/metabolismABSTRACT
The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.