RESUMO
Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.
Assuntos
Região CA1 Hipocampal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Glicosilação , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Transporte Proteico , Receptores de Ácido Caínico/genética , Receptor de GluK2 Cainato , Receptor de GluK3 CainatoRESUMO
Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.
Assuntos
Biomarcadores Tumorais , Proteômica , Proteína EWS de Ligação a RNA , Sarcoma de Células Pequenas , Translocação Genética , Humanos , Proteína EWS de Ligação a RNA/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/patologia , Sarcoma de Células Pequenas/diagnóstico , Feminino , Masculino , Adulto , Adolescente , Adulto Jovem , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/diagnóstico , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/diagnóstico , Criança , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genéticaRESUMO
Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cell-autonomous manner in different cell types. Here, we report an additional non-cell-autonomous function for DR6 in the peripheral nervous system (PNS). DR6-knockout (DR6 KO) mice showed precocious myelination in the PNS Using an in vitro myelination assay, we demonstrate that neuronal DR6 acts in trans on Schwann cells (SCs) and reduces SC proliferation and myelination independently of its cytoplasmic death domain. Mechanistically, DR6 was found to be cleaved in neurons by "a disintegrin and metalloprotease 10" (ADAM10), releasing the soluble DR6 ectodomain (sDR6). Notably, in the in vitro myelination assay, sDR6 was sufficient to rescue the DR6 KO phenotype. Thus, in addition to the cell-autonomous receptor function of full-length DR6, the proteolytically released sDR6 can unexpectedly also act as a paracrine signaling factor in the PNS in a non-cell-autonomous manner during SC proliferation and myelination. This new mode of DR6 signaling will be relevant in future attempts to target DR6 in disease settings.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proliferação de Células , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Células de Schwann/metabolismo , Animais , Morte Celular , Linhagem Celular , Citoplasma/metabolismo , Domínio de Morte , Desintegrinas/metabolismo , Feminino , Células HEK293 , Humanos , Hibridomas , Masculino , Metaloproteases/metabolismo , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Comunicação Parácrina , Fenótipo , Receptores do Fator de Necrose Tumoral/genética , Células de Schwann/ultraestrutura , Especificidade por SubstratoRESUMO
Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Proteínas Reguladoras de Apoptose , Cromatografia Líquida/métodos , Humanos , Espectrometria de Mobilidade Iônica/métodos , Masculino , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
OBJECTIVE: POLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data. METHODS: TCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis. RESULTS: High density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading. CONCLUSIONS: EC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/patologia , Linfócitos do Interstício Tumoral/imunologia , Instabilidade de Microssatélites , Mutação , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/imunologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (Aß) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteômica , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Encéfalo/patologia , Camundongos , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/genéticaRESUMO
Parkin, a RING-between-RING-type E3 ubiquitin ligase associated with Parkinson's disease, has a wide neuroprotective activity, preventing cell death in various stress paradigms. We identified a stress-protective pathway regulated by parkin that links NF-κB signaling and mitochondrial integrity via linear ubiquitination. Under cellular stress, parkin is recruited to the linear ubiquitin assembly complex and increases linear ubiquitination of NF-κB essential modulator (NEMO), which is essential for canonical NF-κB signaling. As a result, the mitochondrial guanosine triphosphatase OPA1 is transcriptionally upregulated via NF-κB-responsive promoter elements for maintenance of mitochondrial integrity and protection from stress-induced cell death. Parkin-induced stress protection is lost in the absence of either NEMO or OPA1, but not in cells defective for the mitophagy pathway. Notably, in parkin-deficient cells linear ubiquitination of NEMO, activation of NF-κB, and upregulation of OPA1 are significantly reduced in response to TNF-α stimulation, supporting the physiological relevance of parkin in regulating this antiapoptotic pathway.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Animais , Apoptose , Fibroblastos/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transdução de Sinais , Transfecção , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased ß2-transferrin levels in patient CSF Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Neurônios/metabolismo , Receptor ErbB-4/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Humanos , Transporte Proteico , Ratos , Receptor ErbB-4/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron-specific. Using a novel hAPP-specific antibody in combination with cell type-specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that-in addition to neurons throughout the brain-astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/metabolismo , Encéfalo/patologia , Fatores Etários , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares , Neurônios/metabolismo , Neurônios/patologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Regulação da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Glicosilação , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Leucemia Linfocítica Crônica de Células B/imunologia , Proteína-Tirosina Quinase ZAP-70/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular , Sobrevivência Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Quinase Syk , Receptor Toll-Like 9/imunologiaRESUMO
Oligomeric assemblies of neurotoxic amyloid beta (Abeta) peptides generated by proteolytical processing of the amyloid precursor protein (APP) play a key role in the pathogenesis of Alzheimer's disease (AD). In recent years, a substantial heterogeneity of Abeta peptides with distinct biophysical and cell biological properties has been demonstrated. Among these, a particularly neurotoxic and disease-specific Abeta variant is N-terminally truncated and modified to pyroglutamate (pE-Abeta). Cell biological and animal experimental studies imply the catalysis of this modification by the enzyme glutaminyl cyclase (QC). However, direct histopathological evidence in transgenic animals from comparative brain region and cell type-specific expression of transgenic hAPP and QC, on the one hand, and on the formation of pE-Abeta aggregates, on the other, is lacking. Here, using single light microscopic, as well as triple immunofluorescent, labeling, we report the deposition of pE-Abeta only in the brain regions of APP-transgenic Tg2576 mice with detectable human APP and endogenous QC expression, such as the hippocampus, piriform cortex, and amygdala. Brain regions showing human APP expression without the concomitant presence of QC (the anterodorsal thalamic nucleus and perifornical nucleus) do not display pE-Abeta plaque formation. However, we also identified brain regions with substantial expression of human APP and QC in the absence of pE-Abeta deposition (the Edinger-Westphal nucleus and locus coeruleus). In these brain regions, the enzymes required to generate N-truncated Abeta peptides as substrates for QC might be lacking. Our observations provide additional evidence for an involvement of QC in AD pathogenesis via QC-catalyzed pE-Abeta formation.
Assuntos
Doença de Alzheimer/metabolismo , Aminoaciltransferases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Doença de Alzheimer/genética , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/genética , Animais , Cabras , Humanos , Imuno-Histoquímica , Camundongos , Modelos Animais , RatosRESUMO
The secretome, the entirety of all soluble proteins either being secreted or proteolytically released by a cell, plays a key role in inter-cellular communication of multi-cellular organisms. Pathological alterations contribute to diseases such as hypertension, cancer, autoimmune disorders or neurodegenerative diseases. Hence, studying disease-related perturbations of the secretome and the secretome itself covers an important aspect of cellular physiology. We recently developed the secretome protein enrichment with click sugars (SPECS) method that enables the analysis of secretomes of in vitro cell cultures even in the presence of FCS with MS. So far, SPECS facilitated the identification of protease substrates of BACE1, SPPL3 and ADAM10. Though, the SPECS method has already enabled deep insights into secretome biology, we aimed to improve the SPECS protocol to obtain even more information from MS-based secretome analysis and reduce the amount of input material. Here, we optimised the reaction buffer, the pH and replaced Dibenzocyclooctyne (DBCO) PEG12-biotin with the more water-soluble variant DBCO-sulpho-biotin to finally provide an optimised protocol of the recently published SPECS protocol. Overall, the number of quantified glycoproteins and their average sequence coverage was increased by 1.6- and 2.4-fold, respectively. Thus, the opzimised SPECS protocol allows reducing the input material by half without losing information. These improvements make the SPECS method more sensitive and more universal applicable to cell types with limited availability.
Assuntos
Química Click/métodos , Glicoproteínas/metabolismo , Proteômica/métodos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Ciclo-Octanos/química , Fibroblastos/metabolismo , Glicoproteínas/análise , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Leucemia Linfocítica Crônica de Células B/metabolismo , CamundongosRESUMO
BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF-APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF-APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation.
Assuntos
Proteínas ADAM/imunologia , Secretases da Proteína Precursora do Amiloide/imunologia , Linfócitos B/imunologia , Ativação Linfocitária , Proteínas de Membrana/imunologia , Esclerose Múltipla/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Autoanticorpos/imunologia , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Esclerose Múltipla/patologia , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1-/- and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1-/- and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1-/- mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.
Assuntos
Secretases da Proteína Precursora do Amiloide/líquido cefalorraquidiano , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/metabolismo , Proteômica/métodos , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Diester Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Signal peptide peptidase-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Complexo de Golgi/metabolismo , Glicosilação , Células HEK293 , HumanosRESUMO
Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Sinapses/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Especificidade por SubstratoRESUMO
Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.
Assuntos
Comunicação Celular , Citosol/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Astrócitos/citologia , Técnicas de Cocultura , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Camundongos , Microscopia Confocal , Doenças Priônicas , Conformação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
Regulated intramembrane proteolysis (RIP) is a critical mechanism for intercellular communication and regulates the function of membrane proteins through sequential proteolysis. RIP typically starts with ectodomain shedding of membrane proteins by extracellular membrane-bound proteases followed by intramembrane proteolysis of the resulting membrane-tethered fragment. However, for the majority of RIP proteases the corresponding substrates and thus, their functions, remain unknown. Proteome-wide identification of RIP protease substrates is possible by mass spectrometry-based quantitative comparison of RIP substrates or their cleavage products between different biological states. However, this requires quantification of peptides from only the ectodomain or cytoplasmic domain. Current analysis software does not allow matching peptides to either domain. Here we present the QARIP (Quantitative Analysis of Regulated Intramembrane Proteolysis) web server which matches identified peptides to the protein transmembrane topology. QARIP allows determination of quantitative ratios separately for the topological domains (cytoplasmic, ectodomain) of a given protein and is thus a powerful tool for quality control, improvement of quantitative ratios and identification of novel substrates in proteomic RIP datasets. To our knowledge, the QARIP web server is the first tool directly addressing the phenomenon of RIP. The web server is available at http://webclu.bio.wzw.tum.de/qarip/. This website is free and open to all users and there is no login requirement.
Assuntos
Proteínas de Membrana/metabolismo , Software , Ácido Aspártico Endopeptidases/metabolismo , Células HEK293 , Humanos , Internet , Espectrometria de Massas , Proteínas de Membrana/química , Peptídeos/análise , Estrutura Terciária de Proteína , Proteólise , ProteômicaRESUMO
The ß-site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) were initially identified as transmembrane aspartyl proteases cleaving the amyloid precursor protein (APP). BACE1 is a major drug target for Alzheimer's disease because BACE1-mediated cleavage of APP is the first step in the generation of the pathogenic amyloid-ß peptides. BACE1, which is highly expressed in the nervous system, is also required for myelination by cleaving neuregulin 1. Several recent proteomic and in vivo studies using BACE1- and BACE2-deficient mice demonstrate a much wider range of physiological substrates and functions for both proteases within and outside of the nervous system. For BACE1 this includes axon guidance, neurogenesis, muscle spindle formation, and neuronal network functions, whereas BACE2 was shown to be involved in pigmentation and pancreatic ß-cell function. This review highlights the recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer's disease. The protease BACE1 is a major drug target in Alzheimer disease. Together with its homolog BACE2, both proteases have an increasing number of functions within and outside of the nervous system. This review highlights recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer disease.