RESUMO
Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.
Assuntos
Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Autofagia/genética , Retículo Endoplasmático/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Estresse do Retículo Endoplasmático , Proteínas de Membrana Transportadoras/metabolismoRESUMO
The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.
Assuntos
Divisão Celular , Proteínas Serina-Treonina Quinases , Neoplasias Uterinas , Animais , Feminino , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Acilação , Divisão Celular/fisiologia , Mutação , Quinase 1 Polo-LikeRESUMO
Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.
Assuntos
Acetilglucosamina , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia Mediada por Chaperonas , Neoplasias de Cabeça e Pescoço , Peptídeos e Proteínas de Sinalização Intracelular , Infecções por Papillomavirus , Proteína Quinase C-alfa , Carcinoma de Células Escamosas de Cabeça e Pescoço , Acetilglucosamina/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Estabilidade Enzimática , Glicosilação , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Infecções por Papillomavirus/metabolismo , Proteína Quinase C-alfa/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologiaRESUMO
M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.
Assuntos
Interleucina-2/genética , Meliteno/genética , Neoplasias Ovarianas/tratamento farmacológico , Pichia/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked ß-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Neoplasias da Mama , Proteínas de Transporte , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas de Ligação a Hormônio da Tireoide , Hormônios Tireóideos , Efeito Warburg em Oncologia , Humanos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Fosforilação , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Feminino , Camundongos , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Acetilglucosamina/metabolismoRESUMO
Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation. Here we investigate how the toxin and antidote products of a wtf-family KMD gene can act antagonistically. Both the toxin and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs PY motifs (Leu/Pro-Pro-X-Tyr) in its N-terminal cytosolic tail to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby preventing it from causing toxicity. A physical interaction between the antidote and the toxin enables the ubiquitinated antidote to translocate the toxin to the endosome and neutralize its toxicity. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs.
Assuntos
Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Antídotos , Ubiquitinação , Complexo de Golgi/metabolismo , Ubiquitina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Lymphocyte infiltration is an important feature of cancer. There is a complex network of chemokines that influence the degree and phenotype of lymphocyte infiltration, as well as the growth, survival, migration, and angiogenesis of tumor cells. High heterogeneity metastasis is a major obstacle to the treatment of breast cancer. Herein, we showed that O-GlcNAcylation of B lymphocyte-induced maturation protein-1 (Blimp-1) in lymphocytes inhibited the migration and invasion of breast cancer cells. It was found that Blimp-1 O-GlcNAcylation at Ser448 and Ser472 in lymphocytes promoted its nuclear localization, and blocked the bindings to three regions upstream of the ccl3l1 promoter to inhibit its expression. Decreased expression of CCL3L1 in lymphocytes not only decreased CCR5 expression in breast cancer cells, but also inhibited the membrane localization and activation of CCR5, thus blocking the migration and invasion of breast cancer cells in vitro. Therefore, O-GlcNAcylation of Blimp-1 in lymphocytes may serve as a new target for the treatment of metastatic breast cancer. IMPLICATIONS: This study reveals a new mechanism by which the lymphatic system promotes breast cancer cell metastasis.
Assuntos
Neoplasias da Mama , Linfócitos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Neoplasias da Mama/patologia , Feminino , Humanos , Linfócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Regiões Promotoras GenéticasRESUMO
Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms-core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both core TRAPP/TRAPPI and Trs120 via its nucleotide-binding domain and binds with Trs31 via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.
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The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.
Assuntos
Exossomos , Melanoma , Animais , Antígeno B7-H1 , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Exossomos/metabolismo , Humanos , Imunoterapia , Camundongos , Fosforilação , Receptor de Morte Celular Programada 1 , Microambiente TumoralRESUMO
High-throughput proteomics based on mass spectrometry (MS) analysis has permeated biomedical science and propelled numerous research projects. pFind 3 is a database search engine for high-speed and in-depth proteomics data analysis. pFind 3 features a swift open search workflow that is adept at uncovering less obvious information such as unexpected modifications or mutations that would have gone unnoticed using a conventional data analysis pipeline. In this protocol, we provide step-by-step instructions to help users mastering various types of data analysis using pFind 3 in conjunction with pParse for data pre-processing and if needed, pQuant for quantitation. This streamlined pParse-pFind-pQuant workflow offers exceptional sensitivity, precision, and speed. It can be easily implemented in any laboratory in need of identifying peptides, proteins, or post-translational modifications, or of quantitation based on 15N-labeling, SILAC-labeling, or TMT/iTRAQ labeling.
RESUMO
[This corrects the article DOI: 10.3892/ol.2015.3125.].
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Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Quinases/metabolismo , Multimerização Proteica/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Fatores de Transcrição/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Cromossômicas não Histona/genética , Fosforilação/genética , Proteínas Quinases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
Assuntos
Microscopia Crioeletrônica/métodos , Exocitose , Saccharomyces cerevisiae/metabolismo , Vesículas Secretórias/metabolismo , Motivos de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Ligação Proteica , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
In the version of this article originally published, the value given for electron dose in Table 1 was incorrect. This value was originally stated as 4.8 but should have been 50. The error has been corrected in the HTML and PDF versions of the article.
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Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.
Assuntos
Reagentes de Ligações Cruzadas/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Escherichia coli/química , Humanos , Espectrometria de Massas , Antígeno Nuclear de Célula em Proliferação/metabolismo , Especificidade por Substrato , Tiorredoxinas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Gene therapy is a promising therapeutic option for the treatment of various cancers, and tumor-targeted plasmids encoding toxic protein genes are potential tools for gene therapy. In the present study, a recombinant plasmid containing the genes for the toxic protein melittin and interleukin-2 (IL-2) was constructed. Melittin and IL-2 are known to play key roles in immunoregulation and cancer therapy, but they each possess defects that limit the clinical application of these proteins. The present study aimed to construct a novel recombinant expression plasmid, pLEGFP-C1-M-IL-2(88Arg, 125Ala), and to improve the biological activity of IL-2 and melittin. The M-IL-2(88Arg, 125Ala) gene was excised from the pPICZαA/M-IL-2(88Arg, 125Ala) plasmid by polymerase chain reaction (PCR). The pLEGFP-C1 plasmid carrying the enhanced green fluorescent protein (EGFP) gene was used as a shuttle plasmid. Subsequent to digestion, the M-IL-2(88Arg, 125Ala) gene was subcloned into the pLEGFP-C1 vector to build the pLEGFP-C1-M-IL-2(88Arg, 125Ala) eukaryotic expression plasmid, which was identified by restriction enzyme digestion and gene sequencing. Confocal microscopy was used to determine the transfection efficiency subsequent to the plasmid being transfected into the cervical cancer HeLa cell line. The cells transfected with the pLEGFP-C1-M-IL-2(88Arg, 125Ala) plasmid demonstrated a decreased transfection efficiency compared with the cells transfected with the pLEGFP-C1 plasmid. The cellular expression of M-IL-2(88Arg, 125Ala) was detected by reverse transcription PCR and western blot analysis. Finally, cell counting kit-8 and apoptosis assays were performed to investigate the effects of the expression of the M-IL-2(88Arg, 125Ala) fusion protein on HeLa cells and to analyze the antitumor activity of the protein. In conclusion, a recombinant eukaryotic pLEGFP-C1-M-IL-2(88Arg, 125Ala) expression plasmid containing the M-IL-2(88Arg, 125Ala) fusion gene was constructed and the M-IL-2(88Arg, 125Ala) fusion protein was successfully expressed in HeLa cells. Furthermore, the M-IL-2(88Arg, 125Ala) fusion protein was able to inhibit HeLa cell proliferation and induce apoptosis in the tumor cells. These findings may offer an alternative method for anticancer therapy. The present study has provided a basis for future studies into the M-IL-2(88Arg, 125Ala) fusion gene.