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1.
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
2.
Commun Biol ; 6(1): 177, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36792683

RESUMO

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.


Assuntos
Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteólise , Domínios Proteicos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Domínio Catalítico
3.
EMBO Mol Med ; 14(10): e16084, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36069059

RESUMO

Fn14 is a cell surface receptor with key functions in tissue homeostasis and injury but is also linked to chronic diseases. Despite its physiological and medical importance, the regulation of Fn14 signaling and turnover is only partly understood. Here, we demonstrate that Fn14 is cleaved within its transmembrane domain by the protease γ-secretase, resulting in secretion of the soluble Fn14 ectodomain (sFn14). Inhibition of γ-secretase in tumor cells reduced sFn14 secretion, increased full-length Fn14 at the cell surface, and enhanced TWEAK ligand-stimulated Fn14 signaling through the NFκB pathway, which led to enhanced release of the cytokine tumor necrosis factor. γ-Secretase-dependent sFn14 release was also detected ex vivo in primary tumor cells from glioblastoma patients, in mouse and human plasma and was strongly reduced in blood from human cancer patients dosed with a γ-secretase inhibitor prior to chimeric antigen receptor (CAR)-T-cell treatment. Taken together, our study demonstrates a novel function for γ-secretase in attenuating TWEAK/Fn14 signaling and suggests the use of sFn14 as an easily measurable pharmacodynamic biomarker to monitor γ-secretase activity in vivo.


Assuntos
Secretases da Proteína Precursora do Amiloide , Receptores de Antígenos Quiméricos , Animais , Biomarcadores , Citocina TWEAK , Humanos , Ligantes , Camundongos , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Fator de Necrose Tumoral alfa
4.
Sci Transl Med ; 13(606)2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380771

RESUMO

Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid ß (Aß). Here, we describe the Uppsala APP mutation (Δ690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) Aß42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing ß-secretase cleavage and affecting α-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated Aß, AßUpp1-42Δ19-24, accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Humanos
5.
Development ; 148(4)2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33531432

RESUMO

KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.


Assuntos
Diferenciação Celular/genética , Movimento Celular/genética , Regulação da Expressão Gênica , Cinesinas/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Processamento Alternativo , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Perfilação da Expressão Gênica , Cinesinas/metabolismo , Camundongos , Mutação , Neurogênese/genética , Proteômica/métodos , Isoformas de RNA , Proteínas Repressoras/metabolismo
6.
EMBO J ; 39(20): e105693, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32954517

RESUMO

To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the "high-performance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Proteômica/métodos , Software , Proteínas ADAM/líquido cefalorraquidiano , Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/líquido cefalorraquidiano , Animais , Antígenos CD/líquido cefalorraquidiano , Antígenos CD/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/líquido cefalorraquidiano , Encéfalo/citologia , Células Cultivadas , Proteínas do Líquido Cefalorraquidiano , Cromatografia Líquida , Ontologia Genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/metabolismo , Análise de Componente Principal , Proteoma/metabolismo , Espectrometria de Massas em Tandem
7.
Semin Cell Dev Biol ; 105: 27-42, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32616437

RESUMO

The intramembrane protease γ-secretase is a hetero-tetrameric protein complex with presenilin as the catalytic subunit and cleaves its membrane protein substrates within their single transmembrane domains. γ-Secretase is well known for its role in Notch signalling and in Alzheimer's disease, where it catalyzes the formation of the pathogenic amyloid ß (Aß) peptide. However, in the 21 years since its discovery many more substrates and substrate candidates of γ-secretase were identified. Although the physiological relevance of the cleavage of many substrates remains to be studied in more detail, the substrates demonstrate a broad role for γ-secretase in embryonic development, adult tissue homeostasis, signal transduction and protein degradation. Consequently, chronic γ-secretase inhibition may cause significant side effects due to inhibition of cleavage of multiple substrates. This review provides a list of 149 γ-secretase substrates identified to date and highlights by which expeirmental approach substrate cleavage was validated. Additionally, the review lists the cleavage sites where they are known and discusses the functional implications of γ-secretase cleavage with a focus on substrates identified in the recent past, such as CHL1, TREM2 and TNFR1. A comparative analysis demonstrates that γ-secretase substrates mostly have a long extracellular domain and require ectodomain shedding before γ-secretase cleavage, but that γ-secretase is also able to cleave naturally short substrates, such as the B cell maturation antigen. Taken together, the list of substrates provides a resource that may help in the future development of drugs inhibiting or modulating γ-secretase activity in a substrate-specific manner.


Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilinas/metabolismo , Humanos , Transdução de Sinais
8.
EMBO J ; 39(15): e103457, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32567721

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 Cainato
10.
eNeuro ; 4(1)2017.
Artigo em Inglês | MEDLINE | ID: mdl-28303257

RESUMO

Calcium signaling controls many key processes in neurons, including gene expression, axon guidance, and synaptic plasticity. In contrast to calcium influx through voltage- or neurotransmitter-gated channels, regulatory pathways that control store-operated calcium entry (SOCE) in neurons are poorly understood. Here, we report a transcriptional control of Stim1 (stromal interaction molecule 1) gene, which is a major sensor of endoplasmic reticulum (ER) calcium levels and a regulator of SOCE. By using a genome-wide chromatin immunoprecipitation and sequencing approach in mice, we find that NEUROD2, a neurogenic transcription factor, binds to an intronic element within the Stim1 gene. We show that NEUROD2 limits Stim1 expression in cortical neurons and consequently fine-tunes the SOCE response upon depletion of ER calcium. Our findings reveal a novel mechanism that regulates neuronal calcium homeostasis during cortical development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Íntrons , Camundongos Endogâmicos BALB C , Proteínas Nucleares , Ligação Proteica , Fatores de Transcrição
11.
BMC Genomics ; 16: 681, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26341353

RESUMO

BACKGROUND: Cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete. RESULTS: In this study, we carried out a ChIP-Seq (chromatin-immunoprecipitation and sequencing) analysis of NEUROD2, an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. Our results suggest that during cortical development NEUROD2 targets key genes that are required for Reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. We also find that NEUROD2 binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons. CONCLUSIONS: Our analysis of in vivo NEUROD2 target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Movimento Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Estudo de Associação Genômica Ampla , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem da Célula/genética , Córtex Cerebral/embriologia , Imunoprecipitação da Cromatina , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Modelos Biológicos , Neuropeptídeos/metabolismo , Ligação Proteica , Proteína Reelina
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