RESUMO
We developed a new cysteine-specific solubilizing tag strategy via a cysteine-conjugated succinimide. This solubilizing tag remains stable under common native chemical ligation conditions and can be efficiently removed with palladium-based catalysts. Utilizing this approach, we synthesized two proteins containing notably difficult peptide segments: interleukin-2 (IL-2) and insulin. This IL-2 chemical synthesis represents the simplest and most efficient approach to date, which is enabled by the cysteine-specific solubilizing tag to synthesize and ligate long peptide segments. Additionally, we synthesized a T8P insulin variant, previously identified in an infant with neonatal diabetes. We show that T8P insulin exhibits reduced bioactivity (a 30-fold decrease compared to standard insulin), potentially contributing to the onset of diabetes in these patients. In summary, our work provides an efficient tool to synthesize challenging proteins and opens new avenues for exploring research directions in understanding their biological functions.
RESUMO
Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , SolubilidadeRESUMO
Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys6 solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.
Assuntos
Ácido GlutâmicoRESUMO
The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.
Assuntos
Peptídeos/química , Subunidades Proteicas/síntese química , Toxina Shiga/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Arginina/química , Cicloexanonas/química , Hidroxilamina/química , Lisina/química , SolubilidadeRESUMO
Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.
Assuntos
Aminas/química , Biotina/química , Corantes Fluorescentes/química , Estreptavidina/química , Anticorpos/química , Anticorpos/imunologia , Biotina/metabolismo , Complexo CD3/imunologia , Citometria de Fluxo , Células HeLa , Humanos , Ligantes , Microscopia Confocal , Mutagênese Sítio-Dirigida , Ligação Proteica , Estabilidade Proteica , Espectrometria de Fluorescência , Estreptavidina/genética , Estreptavidina/metabolismo , Succinimidas/química , TemperaturaRESUMO
The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.
Assuntos
Biologia Computacional/métodos , Proteínas/síntese química , Software , Chaperonina 10/síntese química , Chaperonina 10/química , Chaperonina 60/síntese química , Chaperonina 60/química , Escherichia coli/metabolismo , Proteínas/química , Proteínas Ribossômicas/síntese química , Proteínas Ribossômicas/química , Fator de Necrose Tumoral alfa/síntese química , Fator de Necrose Tumoral alfa/químicaRESUMO
Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences ("helping hands") onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.
Assuntos
Proteínas/química , Proteínas/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética , Fluorenos/química , Dobramento de Proteína , Proteínas Ribossômicas/síntese química , Proteínas Ribossômicas/química , Solubilidade , Proteínas Virais/químicaRESUMO
Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l-TNFα along with the mirror-image d-protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d-peptide with the potential to improve current anti-TNFα therapeutic strategies.
Assuntos
Biblioteca de Peptídeos , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/síntese química , Modelos Moleculares , Estrutura Secundária de Proteína , EstereoisomerismoRESUMO
Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.
Assuntos
Ebolavirus/química , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Sistemas de Liberação de Medicamentos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Proteínas do Envelope Viral/genéticaRESUMO
Mirror-image proteins (composed of D-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger D-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold D-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both L- and D-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both L- and D-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications.
Assuntos
Enzimas/biossíntese , Enzimas/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Aminoácidos/química , Fenômenos Biofísicos , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Enzimas/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroliases/biossíntese , Hidroliases/química , Hidroliases/genética , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Picolínicos/metabolismo , Estrutura Quaternária de Proteína , EstereoisomerismoRESUMO
The adult hippocampus plays a central role in memory formation, synaptic plasticity, and neurogenesis. The subgranular zone of the dentate gyrus contains neural progenitor cells with self-renewal and multilineage potency. Transgene expression of familial Alzheimer's disease-linked mutants of ß-amyloid precursor protein (APP) and presenilin-1 leads to a significant inhibition of neurogenesis, which is potentially linked to age-dependent memory loss. To investigate the effect of neurogenesis on cognitive function in a relevant disease model, FGF2 gene is delivered bilaterally to the hippocampi of APP+presenilin-1 bigenic mice via an adenoassociated virus serotype 2/1 hybrid (AAV2/1-FGF2). Animals injected with AAV2/1-FGF2 at a pre- or postsymptomatic stage show significantly improved spatial learning in the radial arm water maze test. A neuropathological investigation demonstrates that AAV2/1-FGF2 injection enhances the number of doublecortin, BrdU/NeuN, and c-fos-positive cells in the dentate gyrus, and the clearance of fibrillar amyloid-ß peptide (Aß) in the hippocampus. AAV2/1-FGF2 injection also enhances long-term potentiation in another APP mouse model (J20) compared with control AAV2/1-GFP-injected littermates. An in vitro study confirmed the enhanced neurogenesis of mouse neural stem cells by direct AAV2/1-FGF2 infection in an Aß oligomer-sensitive manner. Further, FGF2 enhances Aß phagocytosis in primary cultured microglia, and reduces Aß production from primary cultured neurons after AAV2/1-FGF2 infection. Thus, our data indicate that virus-mediated FGF2 gene delivery has potential as an alternative therapy of Alzheimer's disease and possibly other neurocognitive disorders.
Assuntos
Doença de Alzheimer/metabolismo , Transtornos Cognitivos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipocampo/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células Cultivadas , Transtornos Cognitivos/genética , Transtornos Cognitivos/terapia , Giro Denteado/metabolismo , Giro Denteado/patologia , Dependovirus/genética , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Cytokines play an emerging role as neurotransmitters, neuromodulators, and neurohormones in the brain. This paradigm shift in cytokine function offers a new framework to understand their roles in ameliorating neurodegenerative disorders, such as Alzheimer's disease (AD). Molecular adjuvant therapy of AD animal models with glatiramer acetate induces anti-inflammatory responses and therapeutic effects. Although these effects are potentially mediated through anti-inflammatory cytokine signaling, the exact molecular identities and pathways are poorly understood. Here, we show that virus-mediated expression of the mouse interleukin (IL)-4 gene in beta-amyloid precursor protein + presenilin-1 (APP+PS1) bigenic mice attenuates AD pathogenesis. Introduction of an adeno-associated viral (AAV) vector encoding IL-4 into the hippocampus resulted in sustained expression of IL-4, reduced astro/microgliosis, amyloid-beta peptide (Abeta) oligomerization and deposition, and enhanced neurogenesis. Moreover, increased levels of IL-4 improved spatial learning, promoted phosphorylation of N-methyl-D-aspartate receptor subunit 2B at Tyr 1472, and enhanced its cell surface retention both in vivo and in vitro. Our data suggest that neuronal anti-inflammatory cytokine signaling may be a potential alternative target for non-Abeta-mediated treatment of AD.
Assuntos
Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Sistema Nervoso Central/metabolismo , Interleucina-4/biossíntese , Presenilina-1/genética , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Terapia Genética , Hipocampo/metabolismo , Interleucina-4/genética , Interleucina-4/farmacologia , Camundongos , Camundongos Mutantes , Neurônios/química , Neurônios/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Tau-tubulin kinase-1 (TTBK1) phosphorylates microtubule-associated protein tau at specific serine/threonine residues found in paired helical filaments (PHFs), and its expression is up-regulated in the brain in Alzheimer disease, suggesting its role in tauopathy pathogenesis. To understand the effects of TTBK1 on tauopathy in vivo, we have developed bigenic mice overexpressing full-length TTBK1 and the P301L tau mutant. The bigenic mice show enhanced tau phosphorylation at multiple sites (AT8, 12E8, PHF-1, and pS422), tauC3-immunoreactive tau fragmentation, and accumulation of tau aggregates in cortical and hippocampal neurons at 12-13 mo of age. However, the phosphorylated tau aggregates were predominantly sarkosyl soluble and migrated in the light sucrose density fraction after discontinuous sucrose gradient ultracentrifugation, which suggests that they form small oligomers. The bigenic mice show significant locomotor dysfunction as determined by both rotorod and grip strength tests, as well as enhanced loss of motor neurons in the L4-L5 spinal cord. This neuronal dysfunction and degeneration was associated with increased levels of tau oligomers, cyclin-dependent protein kinase 5 activators p35 and p25, and pY216 phosphorylated glycogen synthase kinase 3-beta. These data suggest that TTBK1 up-regulation enhances tau phosphorylation and oligomerization, whose toxicity results in enhanced neurodegeneration and locomotor dysfunction in a tauopathy animal model.
Assuntos
Mutação , Degeneração Neural/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas tau/metabolismo , Animais , Camundongos , Camundongos Mutantes , Transtornos das Habilidades Motoras/etiologia , Fosforilação , Multimerização Proteica , Tauopatias/etiologia , Regulação para Cima , Proteínas tau/genéticaRESUMO
Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2,100 and 8,800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG)(2)K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and microrheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, time-course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Hidrogéis/química , Hidrogéis/síntese química , Papaína/metabolismo , Peptídeos/química , Polietilenoglicóis/química , Alcinos/química , Materiais Biocompatíveis/metabolismo , Cromatografia em Gel , Elasticidade , Hidrogéis/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Estrutura Molecular , Reologia , Propriedades de Superfície , Água/químicaRESUMO
Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimer's disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Amiloidose/terapia , Quimiocina CCL2/genética , Dependovirus/genética , Gliose/terapia , Aprendizagem em Labirinto/fisiologia , Presenilina-1/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Quimiocina CCL2/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos TransgênicosRESUMO
As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) overexpressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain and proteasome inhibitors significantly accumulated either full-length or processed OTK18 fragments in time- and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.
Assuntos
Calpaína/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Macrófagos/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Adenoviridae/genética , Western Blotting , Calpaína/genética , Linhagem Celular , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Deleção de Genes , Proteínas de Fluorescência Verde , Humanos , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Mutação/genética , Complexo de Endopeptidases do Proteassoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnicas de Cultura de Tecidos , Transfecção , Dedos de Zinco/genéticaRESUMO
Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.