miR-204 functions as a tumor suppressor by regulating SIX1 in NSCLC

The involvement of miR-204 in lung cancer development is unclear. In our study, we analyzed the expression of miR-204 in tumor- and adjacent-tissue samples from 141 patients with non-small cell lung cancer (NSCLC). MiR-204 expression was decreased in tumor samples compared with non-cancerous tissue-derived controls. Moreover, miR-204 expression negatively correlated with homeobox protein SIX1 expression, tumor size and metastasis. MiR-204 silencing in miR-204-positive NSCLC cell lines promoted cell invasion and proliferation. Concomitantly, MiR-204 overexpression resulted in reduced cell proliferation and invasion, upregulated E-cadherin and downregulated N-cadherin and Vimentin expression. SIX1 was identified as a potential target of miR-204, and SIX1 silencing partially compromised the invasive and proliferative capacity of miR-204-deficient cells. Thus, miR-204 may be involved in the NSCLC development.     

A SIX1 degradation inducer blocks excessive proliferation of prostate cancer

Prostate cancer (PC) remains a great medical challenge due to its high incidence and the development of castration resistance in patients treated with androgen deprivation therapy. Deubiquitinases, the enzymes that specifically hydrolyze ubiquitin chains on their substrates, were recently proposed as a serious of critical therapeutic targets for cancer treatment. Our previous study has been reported that the ubiquitin specific peptidase 1 (USP1) functionally acts as a deubiquitinase of sine oculis homeobox homolog 1 (SIX1) and contributes to the proliferation and castration resistance of PC. The stabilization of SIX1 by USP1 partially depends on the status of glucose-regulated protein 75 (GRP75). In this study, we aimed to identify a SIX1 degradation inducer via inhibiting the USP1-SIX1 axis. we screened a range of kinase inhibitors and showed that SNS-032 is the best candidate to trigger the ubiquitinated degradation of SIX1. SNS-032 not only restrains activity of the USP1-SIX1 axis and cell cycle progression, but also results in apoptosis of PC cells. Moreover, the combination of SNS-032 and enzalutamide synergistically induces apoptosis and downregulates expression of USP1, SIX1, and AR/AR-V7 in AR-V7 highly expressed 22Rv1 cells. Overall, our findings may develop a novel and effective strategy to overcome castration resistance in PC for the identification of a SIX1 degradation inducer via targeting the USP1-SIX1 axis.     

Genomic cloning and characterization of the human homeobox gene SIX6 reveals a cluster of SIX genes in chromosome 14 and associates SIX6 hemizygosity with bilateral anophthalmia and pituitary anomalies.

The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several homologues in vertebrates (the SIX gene family). Among them, SIX3 is considered to be the functional orthologue of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications in head development. We report here the cloning and characterization of SIX6, a novel human SIX gene that is the homologue of the chick Six6(Optx2) gene. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 revealed that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3-q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3-q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrated SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders.     

细胞分化抑制因子（Id）研究进展

Id分子(分化抑制因子/DNA结合抑制因子)是一组对碱性螺旋-环-螺旋（bHLH）转录因子活性起负调节作用的转录因子,可抑制细胞分化,促进细胞增殖.哺乳类动物细胞含Id1～Id4 4种Id因子.该分子参与细胞周期调控过程,包括细胞发育、成熟、生长、分化以及死亡等.自1990年发现Id分子以来,有关该分子在基因表达调控、细胞增殖、分化、衰老和肿瘤发生等方面进行了广泛而深入的研究. Id蛋白已成为研究细胞生命过程以及探寻治疗人类疾病有效靶向药物的一类重要分子.     

The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.     

Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development

Hesx1 has been shown to be essential for normal pituitary development. The homeobox gene Six3 is expressed in the developing pituitary gland during mouse development but its function in this tissue has been precluded by the fact that in the Six3-deficient embryos the pituitary gland is not induced. To gain insights into the function of Six3 during pituitary development we have generated Six3^+/−;Hesx1^Cre/+ double heterozygous mice. Strikingly, these mice show marked dwarfism, which is first detectable around weaning, and die by the 5th-6th week of age. Thyroid and gonad development is also impaired in these animals. Analysis of Six3^+/−;Hesx1^Cre/+ compound embryos indicates that hypopituitarism is the likely cause of these defects since pituitary development is severely impaired in these mutants. Similar to the Hesx1-deficient embryos, Rathke's pouch is initially expanded in Six3^+/−;Hesx1^Cre/+ compound embryos due to an increase in cell proliferation. Subsequently, the anterior pituitary gland appears bifurcated, dysmorphic and occasionally ectopically misplaced in the nasopharyngeal cavity, but cell differentiation is unaffected. Our research has revealed a role for Six3 in normal pituitary development, which has likely been conserved during evolution as SIX3 is also expressed in the pituitary gland of the human embryo.     

Functional analysis of the homeodomain protein SIX5

SIX5 (previously known as myotonic dystrophy associated homeodomain protein-DMAHP) is a member of the SIX [sine oculis homeobox (Drosophila) homologue] gene family which encodes proteins containing a SIX domain adjacent to a homeodomain. To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase) and the previously characterised murine Six4 DNA binding site in the Na⁺/K⁺ ATPase α1 subunit gene (ATP1A1) regulatory element (ARE). None of the recombinant proteins showed any affinity for the two putative sites in DMPK. However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with the ARE. The recombinant protein containing both domains formed a second specific complex with the ARE, assumed to be a dimer complex. Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.     

Calcium-induced folding of intrinsically disordered repeat-in-toxin (RTX) motifs via changes of protein charges and oligomerization states

Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by so-called RTX (repeat-in-toxin) motifs found in many virulence factors secreted by numerous gram-negative pathogenic bacteria: RTX proteins are natively disordered in the absence of calcium but fold upon calcium binding. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, contains ～40 RTX motifs organized in five successive blocks separated by non-RTX flanking regions. This RTX domain mediates toxin binding to its eukaryotic cell receptor. We previously showed that the last block of the RTX domain, block V, which is critical for CyaA toxicity, exhibits the hallmarks of intrinsically disordered proteins in the absence of calcium. Moreover, the C-terminal flanking region of CyaA block V is required for its calcium-induced folding. Here, we describe a comprehensive analysis of the hydrodynamic and electrophoretic properties of several block V RTX polypeptides that differ in the presence and/or length of the flanking regions. Our results indicate that the length of the C-terminal flanking region not only controls the calcium-induced folding but also the calcium-induced multimerization of the RTX polypeptides. Moreover, we showed that calcium binding is accompanied by a strong reduction of the net charge of the RTX polypeptides. These data indicate that the disorder-to-order transition in RTX proteins is controlled by a calcium-induced change of the polypeptide charges and stabilized by multimerization.     

Exploring disorder in the human charged biased proteins

A considerable interest has been put in the identification of biased regions in proteins. These regions are frequently associated with a structural role in the cell and particularly with protein disorder. Here, we have investigated the intrinsically disordered regions (IDRs) in the human charged biased proteins identified in our earlier work. We found that 65% of charged biased proteins contained significant IDRs involved particularly in DNA and RNA binding. Also, we have observed that these proteins are well conserved in metazoans and more particularly in mammalian. In addition, the IDRs are located largely in N-terminal, C-terminal sequence flanking the functional domains (FD) and slightly less in (FD) itself. Our work also supports the association between protein disorder and protein–protein/DNA interaction. An example will be described.     

Intrinsically disordered regions in the rodent hepevirus proteome

Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus''s pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.