RESUMO
The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP-competitive mitotic kinesin KIF18A inhibitor BTB-1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB-1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran-binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB-1 greatly improved the viability of IAV-infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.
Assuntos
Resistência à Doença , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Cinesinas/metabolismo , Replicação Viral , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Efeito Citopatogênico Viral , Modelos Animais de Doenças , Resistência à Doença/genética , Expressão Gênica , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Cinesinas/genética , Masculino , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of ß-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-ß1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Sinais de Localização Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/ultraestrutura , alfa Carioferinas/química , alfa Carioferinas/ultraestrutura , Sítios de Ligação , Cristalografia , Ligação Proteica , Conformação ProteicaRESUMO
The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.
Assuntos
Núcleo Celular/metabolismo , Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Transcricional , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Movimento Celular , Proteína de Suscetibilidade a Apoptose Celular/genética , Citoplasma/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosforilação , Transcrição GênicaRESUMO
Background: The purpose of this study was to investigate the potential of plasma cfDNA methylation patterns in reflecting tumour methylation changes, focusing on three candidate sites, cg02469161, cg11528914, and cg20131654. These sites were selected for verification, with a particular emphasis on their association with breast cancer. Methods: We conducted a comprehensive analysis of 850k whole-methylation sequencing data to identify potential markers for breast cancer detection. Subsequently, we investigated the methylation status of the genes Ran-binding protein 3 (RANBP3), Lymphocyte cytoplasmic protein 2 (LCP2), and GRB2 related adaptor protein 2 (GRAP2), situated at the specified sites, using cancer and canceradjacent tissues from 17 breast cancer patients. We also examined the methylation patterns in different molecular subtypes and pathological grades of breast cancer. Additionally, we compared the methylation levels of these genes in plasma cfDNA to their performance in tissues. Results: Our analysis revealed that RANBP3, LCP2, and GRAP2 genes exhibited significant methylation differences between cancer and cancer-adjacent tissues. In breast cancer, these genes displayed diagnostic efficiencies of 91.0%, 90.6%, and 92.2%, respectively. Notably, RANBP3 showed a tendency towards lower methylation in HR+ breast cancer, and LCP2 methylation was correlated with tumour malignancy. Importantly, the methylation levels of these three genes in plasma cfDNA closely mirrored their tissue counterparts, with diagnostic efficiencies of 83.3%, 83.9%, and 77.6% for RANBP3, LCP2, and GRAP2, respectively. Conclusions: Our findings propose that the genes RANBP3, LCP2, and GRAP2, located at the identified methylation sites, hold significant potential as molecular markers in blood for the supplementary diagnosis of breast cancer. This study lays the groundwork for a more in-depth investigation into the changes in gene methylation patterns in circulating free DNA (cfDNA) for the early detection not only of breast cancer but also for various other types of cancer.
RESUMO
Background: Breast cancer developed at a young age (≤45 years) is hypothesized to have unique biology; however, findings in this field are controversial. Methods: We compared the whole transcriptomic profile of young vs. old-age breast cancer using DNA microarray. RNA was extracted from 13 fresh estrogen receptor (ER)-positive primary breast cancer tissues of untreated patients (7 = young age ≤45 years and 6 = old age ≥55 years). In silico validation for the differentially expressed genes (DEGs) by young-age patients was conducted using The Cancer Genome Atlas (TCGA) database. Next, we analyzed the protein expression encoded by two of the significantly down-regulated genes by young-age patients, Glycine N-acyltransferase-like 1 (GLYATL-1) and Ran-binding protein 3 like (RANBP3L), using immunohistochemical analysis in an independent cohort of 56 and 74 ER-positive pre-therapeutic primary breast cancer tissues, respectively. Results: 12 genes were significantly differentially expressed by young-age breast cancers (fold change >2 or <2- with FDR p-value < 0.05). TCGA data confirmed the differential expression of six genes. Protein expression analysis of GLYATL-1 and RANBP3L did not show heterogeneous expression between young and old-age breast cancer tissues. Loss of expression of GLYATL-1 was significantly (p-value 0.005) associated with positive lymph node status. Higher expression of RANBP3L was significantly associated with breast cancers with lower histopathological grades (p-value 0.038). Conclusions: At the transcriptomic level, breast cancer developed in young and old age patients seems homogenous. The variation in the transcriptomic profiles can be attributed to the other clinicopathological characteristics rather than the age of the patient.
RESUMO
Abnormal subcellular localization of proteins is an important cause of tumorigenesis and drug resistance. Chromosome region maintenance 1 (CRM1), the nuclear export regulator of most proteins, has been confirmed to be over-expressed in various malignancies and is regarded as an efficient target. But the potential role of the CRM1 cofactor RanBP3 (Ran Binding Protein 3) is left unrevealed in chronic myeloid leukemia (CML). Here, we first detected the level of RanBP3 in CML and found an elevated RanBP3 expression in CML compared with control. Then we used shRNA lentivirus to down-regulated RanBP3 in imatinib sensitive K562 cells and resistant K562/G01 cells and found RanBP3 silencing inhibited cell proliferation by up-regulating p21, induced caspase3-related cell apoptosis, and enhanced the drug sensitivity of IM in vitro. Notably, we observed that RanBP3 silencing restored imatinib sensitivity of K562 cells in NOD/SCID mice. Mechanistically, the nuclear aggregation of SMAD2/3 revealed that tumor suppressor axis (TGF-ß)-SMAD2/3-p21 was the anti-proliferation program related to RanBP3 knockdown, and the decrease of cytoplasmic ERK1/2 caused by RanBP3 interference leaded to the down-regulation of anti-apoptosis protein p(Ser112)-BAD, which was the mechanism of increased cell apoptosis and enhanced chemosensitivity to imatinib in CML. In summary, this study revealed the expression and potential role of RanBP3 in CML, suggesting that targeting RanBP3 alone or combined with TKIs could improve the clinical response of CML.
RESUMO
Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.