RESUMO
Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, â¼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-ß and α-synuclein.
Assuntos
Amiloidose/metabolismo , Catequina/análogos & derivados , Cadeias Leves de Imunoglobulina/metabolismo , Amiloide/biossíntese , Catequina/farmacologia , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Cadeias Leves de Imunoglobulina/urina , Cinética , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.
Assuntos
Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Fosfatidilserinas/fisiologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Proteínas de Transporte/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transferência de Fosfolipídeos , Ligação Proteica , Eletricidade EstáticaRESUMO
Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that â¼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Insulina/deficiência , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Fragmentos de Peptídeos/metabolismo , Adulto , Animais , Autofagia , Proteínas Contráteis/biossíntese , Proteínas Contráteis/genética , Diabetes Mellitus Experimental/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Peso Molecular , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
BACKGROUND: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions. METHODS: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment. RESULTS: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%). CONCLUSIONS: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration.
Assuntos
Cadeias Leves de Imunoglobulina/sangue , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/sangue , Neoplasia Residual/diagnóstico , Peptídeos/sangue , Medula Óssea/patologia , Exame de Medula Óssea , Regiões Determinantes de Complementaridade/sangue , Regiões Determinantes de Complementaridade/genética , Humanos , Cadeias Leves de Imunoglobulina/genética , Mieloma Múltiplo/patologia , Neoplasia Residual/patologia , Peptídeos/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética , SucçãoRESUMO
Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.
Assuntos
Exossomos/química , Membrana Basal Glomerular/química , Nefropatias/urina , Podócitos/química , Proteinúria/urina , Proteômica/métodos , Urinálise , Urina/química , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Biomarcadores/urina , Estudos de Casos e Controles , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Nefropatias/diagnóstico , Masculino , Dados de Sequência Molecular , Proteinúria/diagnóstico , Adulto JovemRESUMO
Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of â¼3 µM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Multimerização Proteica/fisiologia , Proteínas Repressoras/metabolismo , Calcineurina/química , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Proteínas Interatuantes com Canais de Kv/química , Proteínas Interatuantes com Canais de Kv/genética , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.
Assuntos
Apoptose , Hexoquinase/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ácido Edético/metabolismo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Glicólise , Hexoquinase/genética , Proteínas Interatuantes com Canais de Kv/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , TransfecçãoRESUMO
BACKGROUND: Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation. METHODS: We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides. RESULTS: We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls. CONCLUSIONS: Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1, asporin, cartilage oligomeric matrix protein, chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V, and proprotein convertase subtilisin/kexin 6.
Assuntos
Adenocarcinoma/sangue , Adenocarcinoma/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/metabolismo , Espectrometria de Massas em TandemRESUMO
Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB-deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.
Assuntos
Ciclofilinas/deficiência , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Animais , Tamanho Corporal , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Ciclofilinas/metabolismo , Proteínas da Matriz Extracelular , Células HeLa , Humanos , Cifose/complicações , Cifose/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Chaperonas Moleculares , Osteogênese Imperfeita/complicações , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas/metabolismo , Proteoglicanas/metabolismo , Anormalidades da Pele/patologiaRESUMO
Shotgun proteomics via mass spectrometry (MS) is a powerful technology for biomarker discovery that has the potential to lead to noninvasive disease screening mechanisms. Successful application of MS-based proteomics technologies for biomarker discovery requires accurate expectations of bias, reproducibility, variance, and the true detectable differences in platforms chosen for analyses. Characterization of the variability inherent in MS assays is vital and should affect interpretation of measurements of observed differences in biological samples. Here we describe observed biases, variance structure, and the ability to detect known differences in spike-in data sets for which true relative abundance among defined samples were known and were subsequently measured with the iTRAQ technology on two MS platforms. Global biases were observed within these data sets. Measured variability was a function of mean abundance. Fold changes were biased toward the null and variance of a fold change was a function of protein mass and abundance. The information presented herein will be valuable for experimental design and analysis of the resulting data.
Assuntos
Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Proteômica/métodos , Animais , Bovinos , Galinhas , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Cavalos , Humanos , Análise dos Mínimos Quadrados , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Proteínas/análise , Proteínas/química , Proteômica/normas , Coelhos , Reprodutibilidade dos TestesRESUMO
HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.
Assuntos
Proteínas de Transporte/química , Membrana Celular/química , Proteínas Fúngicas/química , Glicolipídeos/química , Dobramento de Proteína , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Fluorescência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicolipídeos/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Difração de Raios XRESUMO
BACKGROUND: α-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency. METHODS: Serum samples (n = 40) were digested with trypsin, and appropriate ¹³C/¹5N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H2O:acetonitrile:n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 µL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay. RESULTS: For A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry. CONCLUSIONS: The LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency.
Assuntos
alfa 1-Antitripsina/sangue , Alelos , Cromatografia Líquida , Heterozigoto , Homozigoto , Humanos , Imunoensaio , Focalização Isoelétrica , Nefelometria e Turbidimetria , Fenótipo , Espectrometria de Massas em Tandem , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
The clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)-based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens.
Assuntos
Amiloidose/classificação , Microdissecção/métodos , Proteômica/métodos , Feminino , Humanos , Lasers , Masculino , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.
Assuntos
Estrutura Secundária de Proteína/genética , Deficiências na Proteostase/genética , Doença de von Willebrand Tipo 2/genética , Fator de von Willebrand/genética , Substituição de Aminoácidos , Plaquetas/química , Plaquetas/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação com Perda de Função/genética , Espectrometria de Massas , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica/genética , Deficiências na Proteostase/sangue , Deficiências na Proteostase/patologia , Doença de von Willebrand Tipo 2/sangue , Doença de von Willebrand Tipo 2/patologia , Fator de von Willebrand/química , Fator de von Willebrand/ultraestruturaRESUMO
Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex-mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD.
Assuntos
Mesângio Glomerular/química , Glomerulonefrite Membranoproliferativa/patologia , Adolescente , Adulto , Complexo Antígeno-Anticorpo , Biópsia , Estudos de Casos e Controles , Criança , Clusterina/análise , Complemento C3/análise , Complemento C4/análise , Complemento C9/análise , Via Alternativa do Complemento , Mesângio Glomerular/patologia , Humanos , Imunoglobulinas/análise , Espectrometria de Massas , Pessoa de Meia-Idade , Vitronectina/análise , Adulto JovemRESUMO
The concentration range of plasma proteins exceeds the dynamic range of any single analytical method. It has been estimated that the concentration range of serum proteins exceeds ten orders of magnitude (1). Because of this, prior immunoselection of even abundant proteins facilitates the relative nonquantitative observations required to show structural abnormality in primary or in posttranslational structure. Determination of atypical proteins by mass measurement has been reported for genetic defects in glycosylation (2, 3) and for monitoring for transthyretin (TTR) defects (4). Here we describe a rapid method of purification and electrospray introduction of TTR into a mass spectrometer to detect mass changes due to amino acid substitutions. The method currently forms the basis for a clinical assay to ascertain TTR mutations resulting in amyloidosis.
Assuntos
Amiloidose Familiar/sangue , Amiloidose Familiar/diagnóstico , Análise Química do Sangue/métodos , Pré-Albumina/análise , Pré-Albumina/química , Substituição de Aminoácidos , Animais , Humanos , Espectrometria de Massas , Mutação , Pré-Albumina/genética , Pré-Albumina/isolamento & purificação , Controle de Qualidade , CoelhosRESUMO
The c-jun-NH(2)-kinases (JNK) play a critical role in tumor promoter-induced cell transformation and apoptosis. Here, we showed that the nuclear factor of activated T3 (NFAT3) is phosphorylated by JNK1 or JNK2 at Ser(213) and Ser(217), which are located in the conserved SP motif. The transactivation domain of NFAT3 is found between amino acids (aa) 113 and 260 and includes the phosphorylation targets of JNK1 and JNK2. NFAT3 transactivation activity was suppressed in JNK1(-/-) or JNK2(-/-) mouse embryonic fibroblast (MEF) cells compared with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter gene assay indicated that NFAT3 transcriptional activity was increased in a dose-dependent manner by JNK1 or JNK2. Double mutations at Ser(213) and Ser(217) suppressed NFAT3 transactivation activity; and SP600125, a JNK inhibitor, suppressed NFAT3-induced 3xNFAT-luciferase activity. Knockdown of JNK1 or JNK2 suppressed foci formation in NIH3T3 cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 activity and suppressed foci formation. Furthermore, knockdown of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci formation in NIH3T3 cells. Taken together, these results provided direct evidence for the anti-oncogenic potential of the NFAT3 transcription factor.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Humanos , Camundongos , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/genética , Células NIH 3T3 , Fosforilação , Serina/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/antagonistas & inibidores , Ativação TranscricionalRESUMO
Proteinaceous deposits are occasionally encountered in surgically obtained biopsies of the nervous system. Some of these are amyloidomas, although the precise nature of other cases remains uncertain. We studied 13 cases of proteinaceous aggregates in clinical specimens of the nervous system. Proteins contained within laser microdissected areas of interest were identified from tryptic peptide sequences by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Immunohistochemical studies for immunoglobulin heavy and light chains and amyloidogenic proteins were performed in all cases. Histologically, the cases were classified into three groups: 'proteinaceous deposit not otherwise specified' (PDNOS) (n=6), amyloidoma (n=5), or 'intracellular crystals' (n=2). LC-MS/MS demonstrated the presence of lambda, but not kappa, light chain as well as serum amyloid P in all amyloidomas. lambda-Light-chain immunostaining was noted in amyloid (n=5), although demonstrable monotypic lymphoplasmacytic cells were seen in only one case. Conversely, in PDNOS kappa, but not lambda, was evident in five cases, both light chains being present in a single case. In three cases of PDNOS, a low-grade B-cell lymphoma consistent with marginal zone lymphoma was present in the brain specimen (n=2) or spleen (n=1). Lastly, in the 'intracellular crystals' group, the crystals were present within CD68+ macrophages in one case wherein kappa-light chain was found by LC-MS/MS only; the pathology was consistent with crystal-storing histiocytosis. In the second case, the crystals contained immunoglobulin G within CD138+ plasma cells. Our results show that proteinaceous deposits in the nervous system contain immunoglobulin components and LC-MS/MS accurately identifies the content of these deposits in clinical biopsy specimens. LC-MS/MS represents a novel application for characterization of these deposits and is of diagnostic utility in addition to standard immunohistochemical analyses.
Assuntos
Encéfalo/patologia , Proteínas do Tecido Nervoso/química , Nervos Periféricos/patologia , Medula Espinal/patologia , Adulto , Idoso , Feminino , Histiocitose/patologia , Humanos , Cadeias lambda de Imunoglobulina/análise , Linfoma/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Componente Amiloide P Sérico/análise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (muESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Delta 25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Metais/química , Microquímica/métodos , Modelos Químicos , Modelos Moleculares , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrofotometria Ultravioleta/métodos , Sítios de Ligação , Simulação por Computador , Ligação Proteica , Conformação ProteicaRESUMO
Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and ß-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of ß-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a ß-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.