The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

Drosophila STING protein has a role in lipid metabolism.

Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. In this work, we describe a previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage, and downregulated expression of lipid metabolism genes. We found that Drosophila STING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). ACC and FASN also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FASN enzyme activity. Together, our results demonstrate a previously undescribed role of STING in lipid metabolism in Drosophila.

Humanized Drosophila Model of the Meier-Gorlin Syndrome Reveals Conserved and Divergent Features of the Orc6 Protein.

Meier-Gorlin syndrome (MGS) is a rare, autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in genes encoding the subunits of the Origin Recognition Complex (ORC), components of the prereplicative complex and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in the conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, a new mutation in Orc6 was also identified; however, it is localized in the N-terminal domain of the protein. To study the functions of Orc6, we used the human gene to rescue the orc6 deletion in Drosophila Using this "humanized" Orc6-based Drosophila model of MGS, we discovered that unlike the previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. Our studies revealed the importance of evolutionarily conserved and variable domains of Orc6 protein, and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system, as well as provided novel insights into the mechanisms underlying MGS pathology.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

Phosphorylation of Pnut in the Early Stages of Drosophila Embryo Development Affects Association of the Septin Complex with the Membrane and Is Important for Viability.

Septin proteins are polymerizing GTPases that are found in most eukaryotic species. Septins are important for cytokinesis and participate in many processes involving spatial modifications of the cell cortex. In Drosophila, septin proteins Pnut, Sep1, and Sep2 form a hexameric septin complex. Here, we found that septin protein Pnut is phosphorylated during the first 2 hr of Drosophila embryo development. To study the effect of Pnut phosphorylation in a live organism, we created a new Drosophila pnut null mutant that allows for the analysis of Pnut mutations during embryogenesis. To understand the functional significance of Pnut phosphorylation, Drosophila strains carrying nonphosphorylatable and phospho-mimetic mutant pnut transgenes were established. The expression of the nonphosphorylatable Pnut protein resulted in semilethality and abnormal protein localization, whereas the expression of the phospho-mimetic mutant form of Pnut disrupted the assembly of a functional septin complex and septin filament formation in vitro Overall, our findings indicate that the controlled phosphorylation of Pnut plays an important role in regulating septin complex functions during organism development.

Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6.

Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system we introduced MGS mutation in Orc6 and established Drosophila model of MGS. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects. © 2015 Wiley Periodicals, Inc.

Endostatin: A novel inhibitor of androgen receptor function in prostate cancer.

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin-which shares structural similarity with noncanonical nuclear receptor box in AR-antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein-protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

Functional insight into the role of Orc6 in septin complex filament formation in Drosophila.

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.

Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB.

The Origin Recognition Complex (ORC) is a six-subunit protein important for the initiation of DNA replication in eukaryotic cells. Orc6 is the smallest and the least conserved among ORC subunits. It is required for the DNA replication but also has a function in cytokinesis in metazoan species, however, the mechanisms of Orc6 action in these processes are not clear. Here we report a structure of the middle domain of human Orc6. This domain has an overall fold similar to the corresponding helical domain of transcription factor TFIIB. Based on these findings, a model of Orc6 binding to DNA is produced. We have identified amino acids of Orc6 which are directly involved in DNA binding. Alterations of these amino acids abolish DNA binding ability of Orc6 and also result in reduced levels of DNA replication in vitro and in cultured cells. Our data indicate that Orc6 is one of the DNA binding subunits of ORC in metazoan species. We propose that Orc6 may participate in positioning of ORC at the origins of DNA replication similar to the role of TFIIB in positioning transcription preinitiation complex at the promoter.

Study of DNA replication in Drosophila using cell free in vitro system.

Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATP ase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.

Functional analysis of an Orc6 mutant in Drosophila.

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G(1) and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G(1) arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.

The origin recognition complex protein family.

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex.

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster.

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

Multiple functions of the origin recognition complex.

The origin recognition complex (ORC), a heteromeric six-subunit protein, is a central component for eukaryotic DNA replication. The ORC binds to DNA at replication origin sites in an ATP-dependent manner and serves as a scaffold for the assembly of other key initiation factors. Sequence rules for ORC-DNA binding appear to vary widely. In budding yeast the ORC recognizes specific ori elements, however, in higher eukaryotes origin site selection does not appear to depend on the specific DNA sequence. In metazoans, during cell cycle progression, one or more of the ORC subunits can be modified in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled. In addition to its well-documented role in the initiation of DNA replication, the ORC is also involved in other cell functions. Some of these activities directly link cell cycle progression with DNA replication, while other functions seem distinct from replication. The function of ORCs in the establishment of transcriptionally repressed regions is described for many species and may be a conserved feature common for both unicellular eukaryotes and metazoans. ORC subunits were found at centrosomes, at the cell membranes, at the cytokinesis furrows of dividing cells, as well as at the kinetochore. The exact mechanism of these localizations remains to be determined, however, latest results support the idea that ORC proteins participate in multiple aspects of the chromosome inheritance cycle. In this review, we discuss the participation of ORC proteins in various cell functions, in addition to the canonical role of ORC in initiating DNA replication.

A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity.

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis.