RESUMO
Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer's disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.
Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Humanos , Animais , Camundongos , Proteínas tau , Proteínas Amiloidogênicas , Camundongos TransgênicosRESUMO
Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.
RESUMO
The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Nucléolo Celular/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura , Ubiquitina/metabolismoRESUMO
Tau protein accumulation drives toxicity in several neurodegenerative disorders. To better understand the pathways regulating tau homeostasis in disease, we investigated the role of ubiquilins (UBQLNs)-a class of proteins linked to ubiquitin-mediated protein quality control (PQC) and various neurodegenerative diseases-in regulating tau. Cell-based assays identified UBQLN2 as the primary brain-expressed UBQLN to regulate tau. UBQLN2 efficiently lowered wild-type tau levels regardless of aggregation, suggesting that UBQLN2 interacts with and regulates tau protein under normal conditions or early in disease. Moreover, UBQLN2 itself proved to be prone to accumulation as insoluble protein in male and female tau transgenic mice and the human tauopathy progressive supranuclear palsy. Genetic manipulation of UBQLN2 in a tauopathy mouse model demonstrated that a physiological UBQLN2 balance is required for tau homeostasis. UBQLN2 overexpression exacerbated phosphorylated tau pathology and toxicity in mice expressing P301S mutant tau, whereas P301S mice lacking UBQLN2 showed significantly reduced phosphorylated tau. Further studies support the view that an imbalance of UBQLN2 perturbs ubiquitin-dependent PQC and autophagy. We conclude that changes in UBQLN2 levels, whether because of pathogenic mutations or secondary to disease states, such as tauopathy, contribute to proteostatic imbalances that exacerbate neurodegeneration.SIGNIFICANCE STATEMENT We defined a role for the protein quality control protein Ubiquilin-2 (UBQLN2), in age-related neurodegenerative tauopathies. This group of disorders is characterized by the accumulation of tau protein aggregates, which differ when UBQLN2 levels are altered. Given the lack of effective disease-modifying therapies for tauopathies and the function of UBQLN2 in handling various disease-linked proteins, we explored the role of UBQLN2 in regulating tau. We found that UBQLN2 reduced tau levels in cell models but behaved differently in mouse brain, where it accelerated mutant tau pathology and tau-mediated toxicity. A better understanding of the diverse functions of regulatory proteins like UBQLN2 can elucidate some of the causative factors in neurodegenerative disease and outline new routes to therapeutic intervention.
Assuntos
Doenças Neurodegenerativas , Tauopatias , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Tauopatias/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Tauopathies including Alzheimer's disease (AD) are marked by the accumulation of aberrantly modified tau proteins. Acetylated tau, in particular, has recently been implicated in neurodegeneration and cognitive decline. HDAC6 reversibly regulates tau acetylation, but its role in tauopathy progression remains unclear. Here, we identified an HDAC6-chaperone complex that targets aberrantly modified tau. HDAC6 not only deacetylates tau but also suppresses tau hyperphosphorylation within the microtubule-binding region. In neurons and human AD brain, HDAC6 becomes co-aggregated within focal tau swellings and human AD neuritic plaques. Using mass spectrometry, we identify a novel HDAC6-regulated tau acetylation site as a disease specific marker for 3R/4R and 3R tauopathies, supporting uniquely modified tau species in different neurodegenerative disorders. Tau transgenic mice lacking HDAC6 show reduced survival characterized by accelerated tau pathology and cognitive decline. We propose that a HDAC6-dependent surveillance mechanism suppresses toxic tau accumulation, which may protect against the progression of AD and related tauopathies.
Assuntos
Disfunção Cognitiva/patologia , Desacetilase 6 de Histona/metabolismo , Tauopatias/patologia , Proteínas tau/metabolismo , Acetilação , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Disfunção Cognitiva/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Desacetilase 6 de Histona/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fosforilação , Processamento de Proteína Pós-Traducional , Tauopatias/genética , Proteínas tau/genéticaRESUMO
Abnormal intracellular accumulation of aggregated tau is a hallmark feature of Alzheimer's disease and other tauopathies. Pathological tau can undergo a range of post-translational modifications (PTMs) that are implicated as triggers of disease pathology. Recent studies now indicate that tau acetylation, in particular, controls both microtubule binding and tau aggregation, thereby acting as a central regulator of tau's biochemical properties and providing avenues to exploit for potential therapies. Here, using cell-based assays and tau transgenic mice harboring an acetylation-mimic mutation at residue Lys-280 (K280Q), we evaluated whether this substitution modifies the neurodegenerative disease pathology associated with the aggregate-prone tau P301S variant. Strikingly, the addition of a K280Q-substituted variant altered P301S-mediated tau conformation and reduced tau hyperphosphorylation. We further evaluated neurodegeneration markers in K280Q acetylation-mimic mice and observed reduced neuroinflammation as well as restored levels of N-methyl-d-aspartate receptors and post-synaptic markers compared with the parental mice. Thus, substituting a single lysine residue in the context of a P301S disease-linked mutation produces a unique tau species that abrogates some of the cardinal features of tauopathy. The findings of our study indicate that a complex tau PTM code likely regulates tau pathogenesis, highlighting the potential utility of manipulating and detoxifying tau strains through site-specific tau-targeting approaches.
Assuntos
Tauopatias/patologia , Proteínas tau/metabolismo , Acetilação , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Estimativa de Kaplan-Meier , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismo , Tauopatias/metabolismo , Tauopatias/mortalidade , Proteínas tau/genéticaRESUMO
Tau acetylation has recently emerged as a dominant post-translational modification (PTM) in Alzheimer's disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule (MT)-binding region (MTBR), suggesting acetylation could regulate both normal and pathological tau functions. Here, we combined biochemical and cell-based approaches to uncover a dual pathogenic mechanism mediated by tau acetylation. We show that acetylation specifically at residues K280/K281 impairs tau-mediated MT stabilization, and enhances the formation of fibrillar tau aggregates, highlighting both loss and gain of tau function. Full-length acetylation-mimic tau showed increased propensity to undergo seed-dependent aggregation, revealing a potential role for tau acetylation in the propagation of tau pathology. We also demonstrate that methylene blue, a reported tau aggregation inhibitor, modulates tau acetylation, a novel mechanism of action for this class of compounds. Our study identifies a potential "two-hit" mechanism in which tau acetylation disengages tau from MTs and also promotes tau aggregation. Thus, therapeutic approaches to limit tau K280/K281 acetylation could simultaneously restore MT stability and ameliorate tau pathology in AD and related tauopathies.
Assuntos
Processamento de Proteína Pós-Traducional , Tauopatias/metabolismo , Proteínas tau/metabolismo , Acetilação , Animais , Linhagem Celular , Humanos , Camundongos , Microtúbulos/metabolismo , Fosforilação , Agregação Patológica de Proteínas/metabolismo , Tauopatias/patologiaRESUMO
Lysine acetylation has emerged as a dominant post-translational modification (PTM) regulating tau proteins in Alzheimer's disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule-binding region (MTBR), a region that is highly conserved among tau, MAP2, and MAP4 family members, implying that acetylation could represent a conserved regulatory mechanism for MAPs beyond tau. Here, we combined mass spectrometry, biochemical assays, and cell-based approaches to demonstrate that the tau family members MAP2 and MAP4 are also subject to reversible acetylation. We identify a cluster of lysines in the MAP2 and MAP4 MTBR that undergo CBP-catalyzed acetylation, many of which are conserved in tau. Similar to tau, MAP2 acetylation can occur in a cysteine-dependent auto-regulatory manner in the presence of acetyl-CoA. Furthermore, tubulin reduced MAP2 acetylation, suggesting tubulin binding dictates MAP acetylation status. Taken together, these results uncover a striking conservation of MAP2/Tau family post-translational modifications that could expand our understanding of the dynamic mechanisms regulating microtubules.
Assuntos
Lisina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas tau/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Peptídeos/análise , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas tau/químicaRESUMO
The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-). The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine) and diluents (hexanethiol or 2-mercaptoethanol) was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers) in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides.
Assuntos
Eletroquímica/métodos , Ouro/química , Peptídeos/química , Soroalbumina Bovina/isolamento & purificação , Adsorção , Animais , Bovinos , Impedância Elétrica , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/química , Ligação Proteica , Soluções , Propriedades de SuperfícieRESUMO
Tau pathology, including neurofibrillary tangles, develops in Alzheimer's disease (AD). The aggregation and hyperphosphorylation of tau are potential therapeutic targets for AD. Administration of anti-tau antibodies reduces tau pathology in transgenic "tauopathy" mice; however, the optimal tau epitopes and conformations to target are unclear. Also unknown is whether intravenous immunoglobulin (IVIG) products, currently being evaluated in AD trials, exert effects on pathological tau. This study examined the effects of anti-tau antibodies targeting different tau epitopes and the IVIG Gammagard on tau aggregation and preformed tau aggregates. Tau aggregation was assessed by transmission electron microscopy and fluorescence spectroscopy, and the binding affinity of the anti-tau antibodies for tau was evaluated by enzyme-linked immunosorbent assays. Antibodies used were anti-tau 1-150 ("D-8"), anti-tau 259-266 ("Paired-262"), anti-tau 341-360 ("A-10"), and anti-tau 404-441 ("Tau-46"), which bind to tau's N-terminus, microtubule binding domain (MBD) repeat sequences R1 and R4, and the C-terminus, respectively. The antibodies Paired-262 and A-10, but not D-8 and Tau-46, reduced tau fibrillization and degraded preformed tau aggregates, whereas the IVIG reduced tau aggregation but did not alter preformed aggregates. The binding affinities of the antibodies for the epitope for which they were specific did not appear to be related to their effects on tau aggregation. These results confirm that antibody binding to tau's MBD repeat sequences may inhibit tau aggregation and indicate that such antibodies may also degrade preformed tau aggregates. In the presence of anti-tau antibodies, the resulting tau morphologies were antigen-dependent. The results also suggested the possibility of different pathways regulating antibody-mediated inhibition of tau aggregation and antibody-mediated degradation of preformed tau aggregates.
Assuntos
Anticorpos/farmacologia , Imunoglobulinas Intravenosas/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Proteínas tau/ultraestrutura , Anticorpos/imunologia , Humanos , Imunoglobulinas Intravenosas/imunologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/patologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/ultraestrutura , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
A protein-based electrochemical biosensor was developed for detection of tau protein aimed towards electrochemically sensing misfolding proteins. The electrochemical assay monitors tau-tau binding and misfolding during the early stage of tau oligomerization. Electrochemical impedance spectroscopy was used to detect the binding event between solution tau protein and immobilized tau protein (tau-Au), acting as a recognition element. The charge transfer resistance (Rct) of tau-Au was 2.9 ± 0.6 kΩ. Subsequent tau binding to tau-Au decreased the Rct to 0.3 ± 0.1 kΩ (90 ± 3% decrease) upon formation of a tau-tau-Au interface. A linear relationship between the Rct and the solution tau concentration was observed from 0.2 to 1.0 µM. The Rct decrease was attributed to an enhanced charge permeability of the tau-tau-Au surface to a redox probe [Fe(CN)6](3-/4-). The electrochemical and surface characterization data suggested conformational and electrostatic changes induced by tau-tau binding. The protein-based electrochemical platform was highly selective for tau protein over bovine serum albumin and allowed for a rapid sample analysis. The protein-based interface was selective for a non-phosphorylated tau441 isoform over the paired-helical filaments of tau, which were composed of phosphorylated and truncated tau isoforms. The electrochemical approach may find application in screening of the early onset of neurodegeneration and aggregation inhibitors.