RESUMO
Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12-ATG5 conjugate to form an ATG12-ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled-coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle's balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type.
RESUMO
Sept8 is a vesicle associated protein and there are two typical transcriptional variants (Sept8-204 and Sept8-201) expressed in mice brain. Interestingly, the coexpression of Sept8-204/Sept5 induces the formation of small sized vesicle-like structure, while that of the Sept8-201/Sept5 produces large puncta. Sept8 is previously shown to be palmitoylated. Here it was further revealed that protein palmitoylation is required for Sept8-204/Sept5 to maintain small sized vesicle-like structure and colocalize with synaptophysin, since either the expression of nonpalmitoylated Sept8-204 mutant (Sept8-204-3CA) or inhibiting Sept8-204 palmitoylation by 2-BP with Sept5 produces large puncta, which barely colocalizes with synaptophysin (SYP). Moreover, it was shown that the dynamic palmitoylation of Sept8-204 is controlled by ZDHHC17 and PPT1, loss of ZDHHC17 decreases Sept8-204 palmitoylation and induces large puncta, while loss of PPT1 increases Sept8-204 palmitoylation and induces small sized vesicle-like structure. Together, these findings suggest that palmitoylation is essential for the maintenance of the small sized vesicle-like structure for Sept8-204/Sept5, and may hint their important roles in synaptic functions.
Assuntos
Lipoilação , Septinas , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Septinas/genética , Septinas/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
Massive GGGGCC (G4C2) repeat expansion in C9orf72 and the resulting loss of C9orf72 function are the key features of ~50% of inherited amyotrophic lateral sclerosis and frontotemporal dementia cases. However, the biological function of C9orf72 remains unclear. We previously found that C9orf72 can form a stable GTPase activating protein (GAP) complex with SMCR8 (Smith-Magenis chromosome region 8). Herein, we report that the C9orf72-SMCR8 complex is a major negative regulator of primary ciliogenesis, abnormalities in which lead to ciliopathies. Mechanistically, the C9orf72-SMCR8 complex suppresses the primary cilium as a RAB8A GAP. Moreover, based on biochemical analysis, we found that C9orf72 is the RAB8A binding subunit and that SMCR8 is the GAP subunit in the complex. We further found that the C9orf72-SMCR8 complex suppressed the primary cilium in multiple tissues from mice, including but not limited to the brain, kidney, and spleen. Importantly, cells with C9orf72 or SMCR8 knocked out were more sensitive to hedgehog signaling. These results reveal the unexpected impact of C9orf72 on primary ciliogenesis and elucidate the pathogenesis of diseases caused by the loss of C9orf72 function.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Cílios , Demência Frontotemporal , Animais , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Cílios/metabolismo , Expansão das Repetições de DNA , Demência Frontotemporal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Células HEK293RESUMO
Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.
Assuntos
Actinas , Septinas , Animais , Humanos , Camundongos , Actinas/genética , Ansiedade/genética , Neurônios/fisiologia , Pseudópodes/genética , Septinas/genética , Septinas/metabolismo , AprendizagemRESUMO
p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.
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Autofagossomos , Proteínas Ubiquitinadas , Camundongos , Ratos , Humanos , Animais , Autofagossomos/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Autofagia/genética , Acilação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mamíferos/metabolismoRESUMO
Protein S-palmitoylation (hereinafter referred to as protein palmitoylation) is a reversible lipid posttranslational modification catalyzed by the zinc finger DHHC-type containing (ZDHHC) protein family. The reverse reaction, depalmitoylation, is catalyzed by palmitoyl-protein thioesterases (PPTs), including acyl-protein thioesterases (APT1/2), palmitoyl protein thioesterases (PPT1/2), or alpha/beta hydrolase domain-containing protein 17A/B/C (ABHD17A/B/C). Proteins encoded by several oncogenes and tumor suppressors are modified by palmitoylation, which enhances the hydrophobicity of specific protein subdomains, and can confer changes in protein stability, membrane localization, protein-protein interaction, and signal transduction. The importance for protein palmitoylation in tumorigenesis has just started to be elucidated in the past decade; palmitoylation appears to affect key aspects of cancer, including cancer cell proliferation and survival, cell invasion and metastasis, and antitumor immunity. Here we review the current literature on protein palmitoylation in the various cancer types, and discuss the potential of targeting of palmitoylation enzymes or palmitoylated proteins for tumor treatment.
Assuntos
Lipoilação , Neoplasias , Humanos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Especificidade por SubstratoRESUMO
Rationale: Protein palmitoylation is tightly related to tumorigenesis or tumor progression as many oncogenes or tumor suppressors are palmitoylated. AEG-1, an oncogene, is commonly elevated in a variety of human malignancies, including hepatocellular carcinoma (HCC). Although AEG-1 was suggested to be potentially modified by protein palmitoylation, the regulatory roles of AEG-1 palmitoylation in tumor progression of HCC has not been explored. Methods: Techniques as Acyl-RAC assay and point mutation were used to confirm that AEG-1 is indeed palmitoylated. Moreover, biochemical experiments and immunofluorescent microscopy were applied to examine the cellular functions of AEG-1 palmitoylation in several cell lines. Remarkably, genetically modified knock-in (AEG-1-C75A) and knockout (Zdhhc6-KO) mice were established and subjected to the treatment of DEN to induce the HCC mice model, through which the roles of AEG-1 palmitoylation in HCC is directly addressed. Last, HCQ, a chemical compound, was introduced to prove in principal that elevating the level of AEG-1 palmitoylation might benefit the treatment of HCC in xenograft mouse model. Results: We showed that AEG-1 undergoes palmitoylation on a conserved cysteine residue, Cys-75. Blocking AEG-1 palmitoylation exacerbates the progression of DEN-induced HCC in vivo. Moreover, it was demonstrated that AEG-1 palmitoylation is dynamically regulated by zDHHC6 and PPT1/2. Accordingly, suppressing the level of AEG-1 palmitoylation by the deletion of Zdhhc6 reproduces the enhanced tumor-progression phenotype in DEN-induced HCC mouse model. Mechanistically, we showed that AEG-1 palmitoylation adversely regulates its protein stability and weakens AEG-1 and staphylococcal nuclease and tudor domain containing 1 (SND1) interaction, which might contribute to the alterations of the RISC activity and the expression of tumor suppressors. For intervention, HCQ, an inhibitor of PPT1, was applied to augment the level of AEG-1 palmitoylation, which retards the tumor growth of HCC in xenograft model. Conclusion: Our study suggests an unknown mechanism that AEG-1 palmitoylation dynamically manipulates HCC progression and pinpoints that raising AEG-1 palmitoylation might confer beneficial effect on the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Lipoilação , Cisteína/metabolismo , Nuclease do Micrococo/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Endonucleases/metabolismoRESUMO
The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.
Assuntos
Lipoilação , Vesículas Sinápticas , Actinas/metabolismo , Neurônios/metabolismo , Fosforilação , Vesículas Sinápticas/metabolismoRESUMO
Schistosomiasis is caused by parasitic flatworms known as schistosomes and affects over 200 million people worldwide. Prevention of T cell exhaustion by blockade of PD-1 results in clinical benefits to cancer patients and clearance of viral infections, however it remains largely unknown whether loss of PD-1 could prevent or cure schistosomiasis in susceptible mice. In this study, we found that S. japonicum infection dramatically induced PD-1 expression in T cells of the liver where the parasites chronically inhabit and elicit deadly inflammation. Even in mice infected by non-egg-producing unisex parasites, we still observed potent induction of PD-1 in liver T cells of C57BL/6 mice following S. japonicum infection. To determine the function of PD-1 in schistosomiasis, we generated PD-1-deficient mice by CRISPR/Cas9 and found that loss of PD-1 markedly increased T cell count in the liver and spleen of infected mice. IL-4 secreting Th2 cells were significantly decreased in the infected PD-1-deficient mice whereas IFN-γ secreting CD4+ and CD8+ T cells were markedly increased. Surprisingly, such beneficial changes of T cell response did not result in eradication of parasites or in lowering the pathogen burden. In further experiments, we found that loss of PD-1 resulted in both beneficial T cell responses and amplification of regulatory T cells that prevented PD-1-deficient T cells from unleashing anti-parasite activity. Moreover, such PD-1-deficient Tregs exert excessive immunosuppression and express larger amounts of adenosine receptors CD39 and CD73 that are crucial for Treg-mediated immunosuppression. Our experimental results have elucidated the function of PD-1 in schistosomiasis and provide novel insights into prevention and treatment of schistosomiasis on the basis of modulating host adaptive immunity.
Assuntos
Schistosoma japonicum , Esquistossomose Japônica , Animais , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética , Linfócitos T ReguladoresRESUMO
BACKGROUND: Synaptic abnormalities in synaptic proteins are the initial hallmarks of Alzheimer's disease (AD). The higher level of palmitoylation of synaptic proteins was closely associated with amyloid-ß (Aß) in AD. Cattle encephalon glycoside and ignotin (CEGI) have been shown to act as multitarget neurotrophic agents in APPswe/PS1dE9 (APP/PS1) transgenic AD mice. However, it is not clear whether CEGI can influence Aß deposition or whether it does so by the regulation of protein palmitoylation and expression of synaptic proteins in transgenic AD mice. OBJECTIVE: In this study, we investigated the roles of CEGI in modulating postsynaptic density protein 95 (PSD-95) palmitoylation, Aß pathologies, and expression of synaptic-associated proteins in APP/PS1 mice. METHODS: Five-month-old APP/PS1 mice were treated intraperitoneally with 6.6âmL/kg of CEGI for 6 weeks. At the end of the treatment period, APP/PS1 mice were subjected to Morris water maze to test their cognitive functions. Acyl-biotinyl exchange (ABE) for PSD-95 palmitoylation, immunofluorescent staining for expression of PSD-95, N-methyl-D-aspartic acid receptor subunit 2B (NR2B), and synaptotagmin 1 (SYT1) were assessed in mouse brain sections. RESULTS: CEGI treatment in APP/PS1 mice significantly reduced Aß deposition, relieved memory deficits, and decreased PSD-95 palmitoylation while markedly increasing the expression of PSD-95, NR2B, and SYT1 in the frontal cortex. There was a significant correlation between Aß expression and PSD-95 palmitoylation in APP/PS1 mice. CONCLUSION: Our findings demonstrate that CEGI improved AD-like neuropathology, possibly by inhibiting PSD-95 palmitoylation, improving learning memory, and enhancing expression of synaptic-associated proteins, representing a potential therapy for AD treatment.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/complicações , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Bovinos , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/metabolismo , Lobo Frontal/patologia , Glicosídeos , Lipoilação , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Presenilina-1/metabolismoRESUMO
The reversible lipid modification, S-palmitoylation, plays regulatory roles in various physiological processes, e.g., neuronal plasticity and organs development; however, the roles of palmitoylation engaged in testis have yet remained unexplored. Here, we used combined approaches of palm-proteomics, informatics and quantitative PCR to systematically analyze the expression of key enzymes related to protein palmitoylation and identify proteome-wide palmitoylated proteins during the processes of spermatogenesis. Specifically, different timepoints were chosen to collect samples to cover the initiation of meiosis (postnatal, P12), the appearance of the first batch of sperm (P36) and fully fertile status (P60) in mouse. Interestingly, our results showed that only a few enzymes related to protein palmitoylation are highly expressed at later stages (from P36 to P60), rather than in the earlier phase of testis development (P12). To focus on the molecular event of spermatogenesis, we examined the palm-proteomics of testes in P36 and P60 mouse. In total, we identified 4,883 palmitoylated proteins, among which 3,310 proteins match the published palmitoyl-proteome datasets and 1,573 proteins were firstly identified as palmitoylated proteins in this study. Informatics analysis suggested that palmitoylation is involved in events of protein transport, metabolic process, protein folding and cell adhesion, etc. Importantly, further analysis revealed that several networks of palmitoylated proteins are closely associated with sperm morphology and motility. Together, our study laid a solid ground for understanding the roles of protein palmitoylation in spermatogenesis for future studies.
Assuntos
Proteoma , Testículo , Animais , Lipoilação/fisiologia , Masculino , Camundongos , Proteoma/metabolismo , Proteômica/métodos , Sêmen/metabolismo , Testículo/metabolismoRESUMO
Palmitoylation is a special kind of lipid modification that targets proteins to membranes. This protocol introduces the acyl-biotin exchange (ABE) assay to determine the palmitoylation of protein cysteines in yeast. Palmitoylation is exchanged by biotinylated compounds so that the palmitoyl proteins can be affinity-purified for downstream assay by western blot. This protocol is easy to perform and can be applied to other biological sources with slight modifications. This protocol is limited to the detection of cysteine-based palmitoylation. For complete details on the use and execution of this profile, please refer to Lei et al. (2021).
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Lipoilação , Saccharomycetales , Western Blotting , Cisteína/metabolismo , Proteínas/metabolismo , Saccharomycetales/metabolismoRESUMO
Macroautophagy/autophagy is special because the double-layer lipid-formed autophagosome is formed by de novo generation. Phosphatidylinositol-3-phosphate (PtdIns3P) produced by class III phosphatidylinositol 3-kinase complex I (PtdIns3K-CI) is an essential source lipid for the formation of autophagosomes. However, how autophagy is initiated is unknown. In other words, the mechanism by which PtdIns3K-CI is recruited to the phagophore assembly site (PAS) to initiate autophagosome formation is unclear. We recently uncovered the pivotal role of yeast Vac8 in autophagy initiation through the recruitment of PtdIns3K-CI to the PAS. N-terminal palmitoylation of Vac8 anchors it to the vacuole membrane, and the middle ARM domains bind PtdIns3K-CI, leading to the generation of PtdIns3P at the PAS and subsequent autophagosome formation. We found that mouse ARMC3 is the homolog of yeast Vac8 and that its autophagic roles are conserved. Interestingly, spermatids from mice with Armc3 deletion showed blocked ribophagy, low energy levels of mitochondria and motionless flagella, which caused male infertility. These findings revealed a germ tissue-specific autophagic function of ARMC3 in complex eukaryotic species.
Assuntos
Proteínas do Domínio Armadillo , Autofagia , Classe III de Fosfatidilinositol 3-Quinases , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas do Domínio Armadillo/metabolismo , Autofagossomos/metabolismo , Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Masculino , Camundongos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Espermatogênese , Proteínas de Transporte Vesicular/metabolismoRESUMO
How autophagy initiation is regulated and what the functional significance of this regulation is are unknown. Here, we characterized the role of yeast Vac8 in autophagy initiation through recruitment of PIK3C3-C1 to the phagophore assembly site (PAS). This recruitment is dependent on the palmitoylation of Vac8 and on its middle ARM domains for binding PIK3C3-C1. Vac8-mediated anchoring of PIK3C3-C1 promotes PtdIns3P generation at the PAS and recruitment of the PtdIns3P binding protein Atg18-Atg2. The mouse homolog of Vac8, ARMC3, is conserved and functions in autophagy in mouse testes. Mice lacking ARMC3 have normal viability but show complete male infertility. Proteomic analysis indicated that the autophagic degradation of cytosolic ribosomes was blocked in ARMC3-deficient spermatids, which caused low energy levels of mitochondria and motionless flagella. These studies uncovered a function of Vac8/ARMC3 in PtdIns3-kinase anchoring at the PAS and its physical significance in mammalian spermatogenesis with a germ tissue-specific autophagic function.
Assuntos
Autofagia , Ribossomos/metabolismo , Cauda do Espermatozoide/metabolismo , Espermatogênese , Adulto , Animais , Autofagossomos/metabolismo , Células Cultivadas , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Motilidade dos Espermatozoides , Cauda do Espermatozoide/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.
Assuntos
Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Tioléster Hidrolases/genética , Animais , Astrócitos/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Proteína Glial Fibrilar Ácida/genética , Gliose/genética , Humanos , Lipoilação , Camundongos , Camundongos Endogâmicos C57BL , Lipofuscinoses Ceroides Neuronais/genéticaRESUMO
Neutrophils are crucial for immunity and play important roles in inflammatory diseases; however, mouse models selectively deficient in neutrophils are limited, and neutrophil-specific diphtheria toxin (DT)-based depletion system has not yet been established. In this study, we generated a novel knock-in mouse model expressing diphtheria toxin receptor (DTR) under control of the endogenous Ly6G promoter. We showed that DTR expression was restricted to Ly6G+ neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals. We characterized the effects of specific neutrophil depletion in mice at steady-state, with acute inflammation and during tumor growth. Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.
Assuntos
Toxina Diftérica/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/imunologia , Neutrófilos/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologiaRESUMO
BACKGROUND: Astroglioma, one major form of brain tumors, has remained principally tough to handle for decades, due to the complexity of tumor pathology and the poor response to chemo- and radio-therapies. METHODS: Our previous study demonstrated that nifurtimox could regulate the signaling axis of AKT-GSK3ß in various tumor types including the astroglioma U251 cells. Intriguingly, earlier case studies suggested that nifurtimox could possibly permeate the blood brain barrier and arrest neuroblastoma in the brain. These observations jointly encouraged us to explore whether nifurtimox would hinder the growth of astroglioma in vivo. RESULTS: Our results exhibited that nifurtimox could competently hinder the development of astroglioma in the mouse brain as compared to temozolomide, the first line of drug for brain tumors. Meanwhile the surviving rate, as well as the body-weight was dramatically upregulated upon nifurtimox treatment, as compared to that of temozolomide. These findings offered nifurtimox as a better alternative drug in treating astroglioma in vivo. CONCLUSION: Persistently, the manipulation of the signaling axis of AKT-GSK3ß in astroglioma was found in line with earlier findings in neuroblastoma when treated with nifurtimox.
Assuntos
Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Nifurtimox/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Tripanossomicidas/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Zdhhc family genes are composed of 24 members that regulate palmitoylation, a post-translational modification process for proteins. Mutations in genes that alter palmitoylation or de-palmitoylation could result in neurodegenerative diseases and inflammatory disorders. In this study, we found that Zdhhc2 was robustly induced in psoriatic skin and loss of Zdhhc2 in mice by CRISPR/Cas9 dramatically inhibited pathology of the ear skin following imiquimod treatment. As psoriasis is an inflammatory disorder, we analyzed tissue infiltrating immune cells and cytokine production. Strikingly we found that a master psoriatic cytokine interferon-α (IFN-α) in the lesioned skin of wildtype (WT) mice was 23-fold higher than that in Zdhhc2 deficient counterparts. In addition, we found that CD45+ white blood cells (WBC) infiltrating in the skin of Zdhhc2 deficient mice were also significantly reduced. Amelioration in psoriasis and dramatically reduced inflammation of Zdhhc2 deficient mice led us to analyze the cellular components that were affected by loss of Zdhhc2. We found that imiquimod induced plasmacytoid dendritic cell (pDC) accumulation in psoriatic skin, spleen, and draining lymph nodes (DLN) were drastically decreased in Zdhhc2 deficient mice, and the expression of pDC activation marker CD80 also exhibited significantly inhibited in psoriatic skin. In further experiments, we confirmed the cell intrinsic effect of Zdhhc2 on pDCs as we found that loss of zDHHC2 in human CAL-1 pDC dampened both interferon regulatory factor 7 (IRF7) phosphorylation and IFN-α production. Therefore, we identified novel function of Zdhhc2 in controlling inflammatory response in psoriasis in mice and we also confirmed that crucial role of Zdhhc2 in pDCs by regulating IRF7 activity and production of the critical cytokine. Our results finding the dependence of IFN-α production on Zdhhc2 in inflamed murine skin and in human pDCs provide rationale for targeting this new molecule in treatment of inflammation.
Assuntos
Aciltransferases/metabolismo , Células Dendríticas/enzimologia , Interferon-alfa/metabolismo , Psoríase/enzimologia , Pele/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Aciltransferases/genética , Animais , Linhagem Celular , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Imiquimode , Interferon-alfa/genética , Ativação Linfocitária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Psoríase/induzido quimicamente , Psoríase/genética , Psoríase/imunologia , Transdução de Sinais , Pele/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
Neuroblastoma was one of the most life-threatening cancer developed in children, yet the conventional therapies currently used leave an unmet gap for clinical requirements. Temozolomide is the first line of drug in the treatment of neuroblastorma nowadays. Giving the fact that temozolomide treatment offered limited healing effect and patients responded divergently, an alternative beneficial path is urgently requested. Nifurtimox, a drug against Trypanosoma cruzi, was happened to find competent in treating a patient who carried aggressive neuroblastoma. Although in vitro studies demonstrated that nifurtimox has cytotoxic features against tumor cells, a systematic investigation in vivo is generally inadequate. Here we exhibited that nifurtimox could suppress the progression of neuroblastoma in vivo, while maintain the health condition to a great extent. Importantly, as comparing to temozolomide, nifurtimox presented a stronger effect on inhibiting tumor development, strongly suggesting that nifurtimox is a preferential alternative drug in treating neuroblastoma. Additionally, it was shown that Akt-GSK3ß signaling cascade was involved in tumor arrest induced by nifurtimox.