RESUMO
Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation-rich and -poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over-elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that DS over-extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia-dependent signalling.
Assuntos
Cílios/metabolismo , Septinas/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Microtúbulos/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de SinaisRESUMO
The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by auto-inhibition, as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for auto-inhibition of KIF7, whereas the adjacent regulatory coiled coil (rCC) that contributes to auto-inhibition of the motile kinesin-4s KIF21A and KIF21B is not sufficient for KIF7 auto-inhibition. Disease-associated mutations in the inhCC relieve auto-inhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC, and could be increased by disease-associated mutations regardless of the auto-inhibition state of the protein. These findings suggest that loss of KIF7 auto-inhibition and/or altered cilium tip localization can contribute to the pathogenesis of human disease.
Assuntos
Cílios , Cinesinas , Axonema , Proteínas Hedgehog , Humanos , Cinesinas/genética , MicrotúbulosRESUMO
OBJECTIVE: We investigated differentially expressed proteins (DEPs) in human glioblastoma U87 cells after treatment with hederagenin as a therapeutic screening mechanism and provided a theoretical basis for hederagenin in treating glioblastoma. METHODS: The Cell Counting Kit 8 assay was used to analyze the inhibitory effect of hederagenin on the proliferation of U87 cells. Protein was identified by tandem mass tags and LC-MS/MS analysis techniques. Annotation of DEPs, Gene Ontology enrichment and function, and Kyoto Encyclopedia of Genes and Genomes pathways and domains were all examined by bioinformatics. According to the TMT results, hub protein was selected from DEPs for WB verification. RESULTS: Protein quantitative analysis found 6522 proteins in total. Compared with the control group, 43 DEPs (P < 0.05) were involved in the highly enriched signaling pathway in the hederagenin group, among which 20 proteins were upregulated, and 23 proteins were downregulated. These different proteins are mainly involved in the longness regulating pathway-WORM, the hedgehog signaling pathway, Staphylococcus aureus infection, complement, coagulation cascades, and mineral absorption. KIF7 and ATAD2B expression were significantly down-regulated and PHEX and TIMM9 expression were significantly upregulated, according to WB analysis, supporting the TMT findings. CONCLUSION: Hederagenin inhibition of GBM U87 cells may be related to KIF7, which is mainly involved in the hedgehog signaling pathway. Our findings lay a foundation for additional study of the therapeutic mechanism of hederagenin.
RESUMO
Binding of the Hedgehog (Hh) protein signal to its receptor, Patched, induces accumulation of the seven-pass transmembrane protein Smoothened (Smo) within the primary cilium and of the zinc finger transcription factor Gli2 at the ciliary tip, resulting ultimately in Gli-mediated changes in nuclear gene expression. However, the mechanism by which pathway activation is communicated from Smo to Gli2 is not known. In an effort to elucidate this mechanism, we identified Dlg5 (Discs large, homolog 5) in a biochemical screen for proteins that preferentially interact with activated Smo. We found that disruption of Smo-Dlg5 interactions or depletion of endogenous Dlg5 leads to diminished Hh pathway response without a significant impact on Smo ciliary accumulation. We also found that Dlg5 is localized at the basal body, where it associates with another pathway component, Kif7. We show that Dlg5 is required for Hh-induced enrichment of Kif7 and Gli2 at the tip of the cilium but is dispensable for Gpr161 exit from the cilium and the consequent suppression of Gli3 processing into its repressor form. Our findings suggest a bifurcation of Smo activity in Hh response, with a Dlg5-independent arm for suppression of Gli repressor formation and a second arm involving Smo interaction with Dlg5 for Gli activation.
Assuntos
Guanilato Quinases/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Corpos Basais/metabolismo , Linhagem Celular , Cílios/metabolismo , Guanilato Quinases/genética , Células HEK293 , Humanos , Cinesinas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.
Assuntos
Cinesinas , Escoliose , Peixe-Zebra , Animais , Cílios/metabolismo , Humanos , Cinesinas/genética , Mutação , Escoliose/genética , Peixe-Zebra/genética , Proteínas de Peixe-ZebraRESUMO
BACKGROUND: The vertebrate digit pattern is defined by the morphogen Sonic hedgehog (Shh), which controls the activity of Gli transcription factors. Gli1, 2 and 3 are dynamically expressed during patterning. Downstream of Shh, their activity is regulated by Sufu and Kif7, core components of the Shh signaling cascade. The precise roles of these regulators during limb development have not been fully described. We analyze the role of Sufu and Kif7 in the limb and demonstrate that their loss has distinct and synergistic effects on Gli activity and digit pattern. RESULTS: Using a series of mouse mutants, we show that Sufu and Kif7 are expressed throughout limb development and their deletion has distinct effects on Gli levels and limb formation. Concomitant deletion of Sufu and Kif7 results in constitutive pathway activity and severe limb truncation. This is consistent with the recently published two-population model, which suggests that precocious activation of Shh signaling inhibits organizing center formation and limb outgrowth. CONCLUSIONS: Together, our findings demonstrate that perturbations of Sufu and Kif7 affect Gli activity and recapitulate the full spectrum of vertebrate limb defects, ranging from severe truncation to polydactyly.
Assuntos
Padronização Corporal/fisiologia , Proteínas Hedgehog/metabolismo , Membro Posterior/embriologia , Cinesinas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Hedgehog/genética , Cinesinas/genética , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Polidactilia/embriologia , Polidactilia/genética , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. P. aeruginosa secretes a number of toxins by a type III secretion system, and these are important in virulence. One of these toxins, ExoS can induce a cytotoxic effect and is associated with the ability to produce lung damage. ExoS is a bifunctional toxin, with N-terminal GTPase-activating protein (GAP) activity and a C-terminal ADP ribosyl transferase (ADPRT) domain. Although these two domains have numerous potential cellular targets, the overall mechanism of ExoS-induced cytotoxicity remains unclear. We carried out a yeast two-hybrid screen using the ExoS truncation mutant ExoSΔ (residue 1-388), which lacks the 14-3-3 binding site in the ADPRT domain, to identify unknown cellular targets associated with ExoS-induced cytotoxicity. We identified the mammalian factor, kinesin family member 7 (KIF7), which is involved in Hedgehog signaling, as a binding partner for ExoSΔC2. A pull-down assay revealed that ExoS bound to the truncated KIF7 gene encoding the N-terminal domain (residues 1-109) of KIF7. Yeast two-hybrid analysis showed that the ADPRT domain (residues 234-354) of ExoS bound to the truncated KIF7. Furthermore, exoS gene expression and silencing the expression of KIF7 both caused significant cytotoxicity in cultured human bronchial epithelial cells (BEAS-2B). Taken together, our results suggest that ExoS could induce cytotoxicity in BEAS-2B cells by interacting with KIF7.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Cinesinas/metabolismo , Morte Celular/fisiologia , Linhagem Celular , Humanos , Mucosa Respiratória/citologia , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mutations in the KIF7 gene have been implicated in autosomal recessive conditions such as Joubert syndrome, acrocallosal syndrome, and fetal hydrolethalus, as well as in retinal degeneration and other ocular manifestations due to their effect on primary cilia. In this study, we report that the full-field electroretinogram (ERG) test showed non-recordable scotopic ERG responses, while photopic ERG responses were diminished bilaterally. This is a case report of a 62-year-old female patient with painless, progressive vision loss in both eyes. Fundus examination revealed a pale optic nerve head, vessel attenuation, and macular thinning without peripheral pigmentary changes. The full-field electroretinogram (ERG) test showed non-recordable scotopic ERG responses, while photopic ERG responses were diminished bilaterally. Based on these ocular findings, the patient was clinically diagnosed with retinitis pigmentosa (RP) sine pigmento. Genetic testing identified a pathogenic heterozygous mutation in the KIF7 gene with the variant c.61C>T (p.Arg21*). Our case suggests that this pathologic variant may be associated with RP sine pigmento. Further studies are warranted to better understand the role of the KIF7 gene in retinal dystrophies.
RESUMO
Idiopathic scoliosis (IS) is a three-dimensional rotation of the spine >10 degrees with an unknown etiology. Our laboratory established a late-onset IS model in zebrafish (Danio rerio) containing a deletion in kif7. A total of 25% of kif7co63/co63 zebrafish develop spinal curvatures and are otherwise developmentally normal, although the molecular mechanisms underlying the scoliosis are unknown. To define transcripts associated with scoliosis in this model, we performed bulk mRNA sequencing on 6 weeks past fertilization (wpf) kif7co63/co63 zebrafish with and without scoliosis. Additionally, we sequenced kif7co63/co63, kif7co63/+, and AB zebrafish (n = 3 per genotype). Sequencing reads were aligned to the GRCz11 genome and FPKM values were calculated. Differences between groups were calculated for each transcript by the t-test. Principal component analysis showed that transcriptomes clustered by sample age and genotype. kif7 mRNA was mildly reduced in both homozygous and heterozygous zebrafish compared to AB. Sonic hedgehog target genes were upregulated in kif7co63/co63 zebrafish over AB, but no difference was detected between scoliotic and non-scoliotic mutants. The top upregulated genes in scoliotic zebrafish were cytoskeletal keratins. Pankeratin staining of 6 wpf scoliotic and non-scoliotic kif7co63/co63 zebrafish showed increased keratin levels within the zebrafish musculature and intervertebral disc (IVD). Keratins are major components of the embryonic notochord, and aberrant keratin expression has been associated with intervertebral disc degeneration (IVDD) in both zebrafish and humans. The role of increased keratin accumulation as a molecular mechanism associated with the onset of scoliosis warrants further study.
Assuntos
Disco Intervertebral , Escoliose , Animais , Humanos , Escoliose/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Queratinas , Proteínas Hedgehog , Cinesinas/genética , Cinesinas/metabolismoRESUMO
BACKGROUND: The E6 and E7 proteins in human papillomavirus 16 (HPV 16) are the main oncogenes in the occurrence of lung cancer. In recent studies, we found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is still unclear how E6 and E7 regulate LKB1 in lung cancer cells. METHODS: Double directional genetic manipulation and nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well-established lung cancer cell lines. RESULTS: E6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in the cytoplasm only. This suggested that HPV 16 E6 but not E7 downregulates the antitumor activity of LKB1 by downregulating the expression of p-LKB1 in the cytoplasm only. CONCLUSIONS: Here, we demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7. Our findings provide new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggests new therapeutic targets.
Assuntos
Papillomavirus Humano 16/metabolismo , Cinesinas/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Masculino , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , TransfecçãoRESUMO
The correct localization of Hedgehog effectors to the tip of primary cilia is critical for proper signal transduction. The conserved non-motile kinesin Kif7 defines a "cilium-tip compartment" by localizing to the distal ends of axonemal microtubules. How Kif7 recognizes microtubule ends remains unknown. We find that Kif7 preferentially binds GTP-tubulin at microtubule ends over GDP-tubulin in the mature microtubule lattice, and ATP hydrolysis by Kif7 enhances this discrimination. Cryo-electron microscopy (cryo-EM) structures suggest that a rotated microtubule footprint and conformational changes in the ATP-binding pocket underlie Kif7's atypical microtubule-binding properties. Finally, Kif7 not only recognizes but also stabilizes a GTP-form of tubulin to promote its own microtubule-end localization. Thus, unlike the characteristic microtubule-regulated ATPase activity of kinesins, Kif7 modulates the tubulin mechanochemical cycle. We propose that the ubiquitous kinesin fold has been repurposed in Kif7 to facilitate organization of a spatially restricted platform for localization of Hedgehog effectors at the cilium tip.
Assuntos
Cílios/fisiologia , Guanosina Trifosfato/metabolismo , Cinesinas/metabolismo , Mecanotransdução Celular , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Humanos , Cinesinas/química , Cinesinas/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transdução de Sinais , Tubulina (Proteína)/genéticaRESUMO
This study investigated kinesin family member 7 (KIF7) expression and function in prostate cancer (PCa). Our results showed that KIF7 was significantly downregulated in PCa, compared with normal, benign prostatic hyperplasia and prostate intraepithelial neoplasia tissues, partially through promoter hypermethylation. We further investigated the effects of KIF7 coiled coil (CC) domain and motor domain (MD) on PCa development in vitro and in vivo. Our results showed that KIF7-CC but not KIF7-MD significantly attenuated proliferation and colony formation, impeded migration and invasion, induced apoptosis and sensitized PCa cells to paclitaxel. Further analysis revealed that KIF7-CC enhanced LKB1 expression and phosphorylation at Ser428, which induced PTEN phosphorylation at Ser380/Thr382/383 and consequently blocked AKT phosphorylation at Ser473. Downregulation of LKB1 significantly attenuated the suppressive effects of KIF7-CC on cell proliferation, colony formation and AKT phosphorylation. Furthermore, our in vivo studies showed that KIF7-CC reduced prostate tumorigenesis in cell-derived xenografts. Downregulation of LKB1 abrogated the anti-tumor effects of KIF7-CC in these xenografts. Taken together, these findings provide the first evidence to support the role of KIF7 as a negative regulator that inhibits PCa development partially through LKB1-mediated AKT inhibition.
RESUMO
Kif7 is a ciliary kinesin motor protein that regulates mammalian Hedgehog pathway activation through influencing structure of the primary cilium. Here we show that Kif7 is required for normal T-cell development, despite the fact that T-cells lack primary cilia. Analysis of Kif7-deficient thymus showed that Kif7-deficiency increases the early CD44+CD25+CD4-CD8- thymocyte progenitor population but reduces differentiation to CD4+CD8+ double positive (DP) cell. At the transition from DP to mature T-cell, Kif7-deficiency selectively delayed maturation to the CD8 lineage. Expression of CD5, which correlates with TCR signal strength, was reduced on DP and mature CD4 and CD8 cells, as a result of thymocyte-intrinsic Kif7-deficiency, and Kif7-deficient T-cells from radiation chimeras activated less efficiently when stimulated with anti-CD3 and anti-CD28 in vitro. Kif7-deficient thymocytes showed higher expression of the Hedgehog target gene Ptch1 than WT, but were less sensitive to treatment with recombinant Shh, and Kif7-deficient T-cell development was refractory to neutralisation of endogenous Hh proteins, indicating that Kif7-deficient thymocytes were unable to interpret changes in the Hedgehog signal. In addition, Kif7-deficiency reduced cell-surface MHCII expression on thymic epithelial cells.
Assuntos
Diferenciação Celular/genética , Células Epiteliais/metabolismo , Cinesinas/genética , Complexo Principal de Histocompatibilidade/genética , Timócitos/citologia , Timócitos/metabolismo , Timo/fisiologia , Animais , Biomarcadores , Expressão Gênica , Genótipo , Proteínas Hedgehog/metabolismo , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Knockout , Fenótipo , Transdução de Sinais , Timócitos/imunologiaRESUMO
PURPOSE: The aim of this study was to use the TaqMan OpenArray system to evaluate associations between 39 genes and the etiology of nonsyndromic cleft lip and palate (NSCLP) in a Brazilian population. MATERIAL AND METHODS: This case-control association study was designed with 80.11% statistical power according to logistic regression (GPOWER software). The case group had 182 patients with NSCLP enrolled in the Brazilian Database on Orofacial Clefts. The controls included 355 healthy individuals with no history of oral clefting in the past three generations. All samples were genotyped for 253 tag single nucleotide polymorphisms (tagSNPs) in 39 genes, including two that had recently been associated with this process. The association analysis was performed using logistic regression and stepwise regression. The results were corrected for multiple testing [Bonferroni correction and False Discovery Rate (FDR)]. RESULTS: Twenty-four SNPs in 16 genes were significantly associated with the etiology of NSCLP, including MSX1, SPRY1, MSX2, PRSS35, TFAP2A, SHH, VAX1, TBX10, WNT11, PAX9, BMP4, JAG2, AXIN2, DVL2, KIF7, and TCBE3. Stepwise regression analysis revealed that 11 genes contributed to 15.5% of the etiology of NSCLP in the sample. CONCLUSION: This is the first study to associate KIF7 and TCEB3 with the etiology of NSCLP. New technological approaches using the same design should help to identify further etiological susceptibility variants.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo de Nucleotídeo Único , Brasil , Estudos de Casos e Controles , Elonguina , Feminino , Humanos , Cinesinas/genética , Masculino , Fatores de Transcrição/genéticaRESUMO
Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. Mechanisms whereby T-box3 regulates limb development are poorly understood. We discovered requirements for T-box at multiple stages of mouse limb development and distinct molecular functions in different tissue compartments. Early loss of T-box3 disrupts limb initiation, causing limb defects that phenocopy Sonic Hedgehog (Shh) mutants. Later ablation of T-box3 in posterior limb mesenchyme causes digit loss. In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor. Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.
Assuntos
Membro Anterior/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/embriologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Membro Anterior/anormalidades , Membro Posterior/anormalidades , Cinesinas/metabolismo , Camundongos , Mapeamento de Interação de Proteínas , Proteínas com Domínio T/genética , Proteína Gli3 com Dedos de ZincoRESUMO
Acrocallosal syndrome (ACLS) is a rare autosomal recessive disorder characterized by agenesis of the corpus callosum, facial dysmorphism, postaxial polydactyly of the hands as well as preaxial polydactyly of the feet, and developmental delay. Mutations in the KIF7 gene, encoding a molecule within the Sonic hedgehog (SHH) pathway, have been identified as causative for ACLS but also for the fatal hydrolethalus syndrome and some cases of Joubert syndrome. We report here on a Tunisian boy who shows the clinical characteristics of ACLS and was found to have a novel homozygous KIF7 nonsense mutation. Further, we summarize the current knowledge about the clinical spectrum associated with KIF7 mutations as well as genetic and/or phenotypic overlap with ciliopathies and other mutations in the SHH pathway.