Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 146
Filtrar
1.
Nat Commun ; 15(1): 5944, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39013852

RESUMO

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Animais , Humanos , Camundongos , Conformação Proteica , Multimerização Proteica , Células HEK293 , Encéfalo/metabolismo , Encéfalo/patologia , Mutação , Mutação com Perda de Função
2.
J Clin Invest ; 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38888964

RESUMO

The ß-secretase BACE1 is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet, little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, non-human primates and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for a safer prevention of Alzheimer's disease.

3.
Cell Mol Life Sci ; 81(1): 227, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38775843

RESUMO

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.


Assuntos
Autofagia , Catepsinas , Lisossomos , Proteólise , Humanos , Lisossomos/metabolismo , Catepsinas/metabolismo , Catepsinas/genética , Células HeLa , Endocitose , Catepsina L/metabolismo , Catepsina L/genética , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética
4.
Cell Mol Life Sci ; 81(1): 163, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38570362

RESUMO

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.


Assuntos
Proteínas de Transporte , Infecções por Vírus Epstein-Barr , Animais , Humanos , Camundongos , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Proteínas de Transporte/metabolismo , Herpesvirus Humano 4 , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout
5.
Front Med (Lausanne) ; 11: 1345310, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38646559

RESUMO

Background: The aim of the study was to determine the impact that PHTLS® course participation had on self-confidence of emergency personnel, regarding the pre-hospital treatment of patients who had suffered severe trauma. Furthermore, the goal was to determine the impact of specific medical profession, work experience and prior course participation had on the benefits of PHTLS® training. Methods: A structured questionnaire study was performed. Healthcare providers from local emergency services involved in pre-hospital care in the metropolitan area of Zurich (Switzerland, Europe) who completed a PHTLS® course were included. Altered self-confidence, communication, and routines in the treatment of severe trauma patients were examined. The impact of prior course participation, work experience and profession on course benefits were evaluated. Results: The response rate was 76%. A total of 6 transport paramedics (TPs), 66 emergency paramedics (EPs) and 15 emergency doctors (EDs) were included. Emergency paramedics had significantly more work experience compared with EDs (respectively 7.1 ± 5.7 yrs. vs. 4.5 ± 2.1 yrs., p = 0.004). 86% of the participants reported increased self-confidence in the pre-hospital management of severe trauma upon PHTLS® training completion. Moreover, according to 84% of respondents, extramural treatment of trauma changed upon course completion. PHTLS® course participants had improved communication in 93% of cases. This was significantly more frequent in EPs than TPs (p = 0.03). Multivariable analysis revealed emergency paramedics benefit the most from PHTLS® course participation. Conclusion: The current study shows that PHTLS® training is associated with improved self-confidence and enhanced communication, with regards to treatment of severe trauma patients in a pre-hospital setting, among medical emergency personnel. Additionally, emergency paramedics who took the PHTLS® course improved in overall self-confidence. These findings imply that all medical personal involved in the pre-hospital care of trauma patients, in a metropolitan area in Europe, do benefit from PHTLS® training. This was independent of the profession, previous working experience or prior alternative course participation.

6.
Nat Aging ; 4(4): 595-612, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38519806

RESUMO

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.


Assuntos
Células Endoteliais , Proteômica , Camundongos , Animais , Células Endoteliais/metabolismo , Proteômica/métodos , Encéfalo/metabolismo , Endotélio/metabolismo , Apolipoproteínas E/metabolismo
7.
Int J Mol Sci ; 25(3)2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38339197

RESUMO

Alterations in the gut microbiome are associated with the pathogenesis of Alzheimer's disease (AD) and can be used as a diagnostic measure. However, longitudinal data of the gut microbiome and knowledge about its prognostic significance for the development and progression of AD are limited. The aim of the present study was to develop a reliable predictive model based on gut microbiome data for AD development. In this longitudinal study, we investigated the intestinal microbiome in 49 mild cognitive impairment (MCI) patients over a mean (SD) follow-up of 3.7 (0.6) years, using shotgun metagenomics. At the end of the 4-year follow-up (4yFU), 27 MCI patients converted to AD dementia and 22 MCI patients remained stable. The best taxonomic model for the discrimination of AD dementia converters from stable MCI patients included 24 genera, yielding an area under the receiver operating characteristic curve (AUROC) of 0.87 at BL, 0.92 at 1yFU and 0.95 at 4yFU. The best models with functional data were obtained via analyzing 25 GO (Gene Ontology) features with an AUROC of 0.87 at BL, 0.85 at 1yFU and 0.81 at 4yFU and 33 KO [Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog] features with an AUROC of 0.79 at BL, 0.88 at 1yFU and 0.82 at 4yFU. Using ensemble learning for these three models, including a clinical model with the four parameters of age, gender, body mass index (BMI) and Apolipoprotein E (ApoE) genotype, yielded an AUROC of 0.96 at BL, 0.96 at 1yFU and 0.97 at 4yFU. In conclusion, we identified novel and timely stable gut microbiome algorithms that accurately predict progression to AD dementia in individuals with MCI over a 4yFU period.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Microbioma Gastrointestinal , Humanos , Doença de Alzheimer/genética , Microbioma Gastrointestinal/genética , Estudos Longitudinais , Prognóstico , Disfunção Cognitiva/etiologia , Progressão da Doença , Biomarcadores
8.
FASEB J ; 38(2): e23442, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38275103

RESUMO

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.


Assuntos
Secretases da Proteína Precursora do Amiloide , Lipoilação , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Precursor de Proteína beta-Amiloide/metabolismo
9.
J Proteome Res ; 23(1): 117-129, 2024 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-38015820

RESUMO

The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.


Assuntos
Plasma , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Fluxo de Trabalho , Reprodutibilidade dos Testes , Plasma/química
10.
Mol Neurodegener ; 18(1): 70, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37775827

RESUMO

BACKGROUND: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. METHODS: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study. RESULTS: We identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer's (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. CONCLUSIONS: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer's Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.


Assuntos
Doença de Alzheimer , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Amiloidogênicas/metabolismo , Biomarcadores/metabolismo , Meios de Cultivo Condicionados/farmacologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Granulinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Knockout , Microglia/metabolismo , Proteoma , Proteômica
11.
Nat Commun ; 14(1): 5034, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37596282

RESUMO

Prion-like spreading of protein misfolding is a characteristic of neurodegenerative diseases, but the exact mechanisms of intercellular protein aggregate dissemination remain unresolved. Evidence accumulates that endogenous retroviruses, remnants of viral germline infections that are normally epigenetically silenced, become upregulated in neurodegenerative diseases such as amyotrophic lateral sclerosis and tauopathies. Here we uncover that activation of endogenous retroviruses affects prion-like spreading of proteopathic seeds. We show that upregulation of endogenous retroviruses drastically increases the dissemination of protein aggregates between cells in culture, a process that can be inhibited by targeting the viral envelope protein or viral protein processing. Human endogenous retrovirus envelopes of four different clades also elevate intercellular spreading of proteopathic seeds, including pathological Tau. Our data support a role of endogenous retroviruses in protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading.


Assuntos
Esclerose Lateral Amiotrófica , Retrovirus Endógenos , Príons , Humanos , Retrovirus Endógenos/genética , Agregados Proteicos , Antivirais
12.
bioRxiv ; 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37398209

RESUMO

Background: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. Methods: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), which were genetically modified to yield the most opposite homeostatic ( TREM2- knockout) and disease-associated ( GRN -knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify changes in microglial and cerebrospinal (CSF) proteome of Grn - and Trem2 -knockout mice. Additionally, we analyzed the proteome of GRN - and TREM2 -knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort with 11 GRN mutation carriers and 12 non-carriers, as well as the proteomic data set available from the European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD). Findings: We identified proteomic changes between the opposite activation states in mouse microglia and cerebrospinal fluid (CSF), as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of AD patients. In AD, these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. Interpretation: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these marker proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the DIAN (Dominantly Inherited Alzheimer's Disease Network) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198 to CH, SFL and DP) and a Koselleck Project HA1737/16-1 (to CH).

13.
Nat Metab ; 5(8): 1364-1381, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37430025

RESUMO

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.


Assuntos
Esclerose Múltipla , Doenças Neuroinflamatórias , Animais , Camundongos , Axônios/patologia , Esclerose Múltipla/patologia , Neurônios/patologia , Inflamação/patologia
14.
Mol Nutr Food Res ; 67(18): e2300137, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37465844

RESUMO

SCOPE: Maillard reaction products (MRPs) are believed to interact with the receptor for advanced glycation endproducts (RAGE) and lead to a pro-inflammatory cellular response. The structural basis for this interaction is scarcely understood. This study investigates the effect of individual lysine modifications in free form or bound to casein on human colon cancer cells. METHODS AND RESULTS: Selectively glycated casein containing either protein-bound N-ε-carboxymethyllysine (CML), N-ε-fructosyllysine (FL), or pyrraline is prepared and up to 94%, 97%, and 61% of lysine modification could be attributed to CML, FL, or pyrraline, respectively. HCT 116 cells are treated with free CML, pyrraline, FL, or modified casein for 24 h. Native casein is used as control. Intracellular MRP content is analyzed by UPLC-MS/MS. Microscopic analysis of the transcription factors shows no activation of NFκB by free or protein-bound FL or CML, whereas casein containing protein-bound pyrraline activates Nrf2. RAGE expression is not influenced by free or casein-bound MRPs. Activation of Nrf2 by pyrraline-modified casein is confirmed by analyzing Nrf2 target proteins NAD(P)H dehydrogenase (quinone 1) (NQO1) and heme oxygenase-1 (HO-1). CONCLUSION: Studies on the biological effects of glycated proteins require an individual consideration of defined structures. General statements on the effect of "AGEs" in biological systems are scientifically unsound.


Assuntos
Lisina , Reação de Maillard , Humanos , Lisina/metabolismo , Fator 2 Relacionado a NF-E2 , Caseínas/química , Cromatografia Líquida , Receptor para Produtos Finais de Glicação Avançada , Células HCT116 , Espectrometria de Massas em Tandem , Produtos Finais de Glicação Avançada/química
15.
Front Mol Biosci ; 10: 1162504, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37388246

RESUMO

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used "surface-spanning enrichment with click-sugars (SUSPECS)" proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding.

16.
EBioMedicine ; 89: 104456, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36745974

RESUMO

A major evolution from purely clinical diagnoses to biomarker supported clinical diagnosing has been occurring over the past years in neurology. High-throughput methods, such as next-generation sequencing and mass spectrometry-based proteomics along with improved neuroimaging methods, are accelerating this development. This calls for a consensus framework that is broadly applicable and provides a spot-on overview of the clinical validity of novel biomarkers. We propose a harmonized terminology and a uniform concept that stratifies biomarkers according to clinical context of use and evidence levels, adapted from existing frameworks in oncology with a strong focus on (epi)genetic markers and treatment context. We demonstrate that this framework allows for a consistent assessment of clinical validity across disease entities and that sufficient evidence for many clinical applications of protein biomarkers is lacking. Our framework may help to identify promising biomarker candidates and classify their applications by clinical context, aiming for routine clinical use of (protein) biomarkers in neurology.


Assuntos
Doenças do Sistema Nervoso , Humanos , Biomarcadores , Proteômica/métodos , Espectrometria de Massas , Neuroimagem
17.
Mol Neurodegener ; 18(1): 13, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36810097

RESUMO

BACKGROUND: The protease BACE1 is a major drug target for Alzheimer's disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates. METHODS: To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors. RESULTS: Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal. CONCLUSION: BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.


Assuntos
Doença de Alzheimer , Camundongos , Humanos , Animais , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide , Receptor gp130 de Citocina/uso terapêutico , Ácido Aspártico Endopeptidases , Interleucina-6 , Proteínas do Tecido Nervoso
18.
Herzschrittmacherther Elektrophysiol ; 34(1): 19-25, 2023 Mar.
Artigo em Alemão | MEDLINE | ID: mdl-36692539

RESUMO

Exercise testing is an essential part of the annual sports medical check-up for competitive athletes. The aim of the examination is to assess the current health status and to perform medical clearance for competitive sports. In addition, assessment of intraindividual changes over time, interindividual comparisons, and determination of training recommendations are of interest. For the assessment of cardiopulmonary fitness, a bicycle or treadmill ergometry with ECG recordings is usually conducted. The exercise test is performed according to standardized exercise protocols until maximum individual exhaustion. In competitive sports, understanding of the muscular metabolic situation during exercise is crucial for assessing current exercise capacity and for managing workouts. Therefore, exercise tests are often performed as lactate performance testing or cardiopulmonary exercise test. Lactate performance testing is one of the most frequently used methods regarding exercise testing in competitive sports and is a fixed component in most of the annual medical check-ups, which are performed in the sports medicine examination centers of the German Olympic Sports Confederation (DOSB). It enables the analysis of aerobic and anaerobic metabolism during exercise, which allows identification of individual endurance training intensities based on the underlying metabolism. Cardiopulmonary exercise testing is also suitable to determine the maximum oxygen uptake and to better understand medical issues related to exercise capacity.


Assuntos
Teste de Esforço , Consumo de Oxigênio , Humanos , Teste de Esforço/métodos , Oxigênio , Atletas , Ácido Láctico
19.
Cell ; 185(26): 5040-5058.e19, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36563667

RESUMO

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.


Assuntos
Doença de Alzheimer , Proteoma , Camundongos , Humanos , Animais , Proteoma/análise , Proteômica/métodos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Espectrometria de Massas , Placa Amiloide
20.
Sci Rep ; 12(1): 20987, 2022 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-36470941

RESUMO

Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate's ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases.


Assuntos
Ácido Aspártico Endopeptidases , Proteínas de Membrana , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexo de Golgi/metabolismo , Glicosilação , Polissacarídeos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...