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    ABSTRACT: Mapping sites of wild-type SUMO modification is a challenging endeavour. Here we postulate that a combination of chemical derivatistation and collision-induced dissociation (CID) could be used to generate SUMO remnant diagnostic ions to aid both detection of these isopeptides and increase the analytical value of the product ion spectra required to characterize the nature and position of modification. SUMO(2/3)ylated proteins were digested with trypsin to generate isopeptides bearing TGG and QTGG isotags. The resulting digests were then dimethyl labelled followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) utilising CID in a data-dependent acquisition on a QSTAR XL. Product ion spectra were interrogated for the presence of iso-N-terminal fragment ions in addition to backbone sequence ions. The ability to diagnostically detect these isopeptides was tested by generation of co-XICs of the iso-N-terminal fragments in a semi-complex background. Dimethyl labelling facilitated the robust detection of a1', b2' & b3' (TGG isotag) and a1', b2' & b4' (QTGG isotag) ions. The abundance of both N-terminal and iso-N-terminal fragment ions, supported by dimethyl labelling, facilitated the generation of information-rich product ion spectra of these isopeptides to aid confident site assignment. Moreover, the diagnostic nature of the combined XICs of the iso-N-terminal fragments supported detection of the isopeptide signals from a semi-complex background. A combination of dimethyl labelling and CID does indeed lead to the generation of SUMO remnant isopeptide product ion spectra which are more analytically rich. This enables an improvement in characterization of both the isotag and backbone sequences and the site of modification. The diagnostic value of iso-N-terminal fragment ions allows for post-acquisition XIC interrogation to detect putative isopeptides of interest. Copyright © 2013 John Wiley & Sons, Ltd.
    Rapid Communications in Mass Spectrometry 09/2013; 27(18):2108-14.
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    ABSTRACT: DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.
    Cell Reports 03/2013;
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    ABSTRACT: BACKGROUND: The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation. RESULTS: Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol ε (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol ε to interact with the replisome after initiation. CONCLUSIONS: These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.
    Current biology: CB 03/2013;
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    ABSTRACT: Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. Figure ᅟ
    Journal of the American Society for Mass Spectrometry 01/2013;
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    ABSTRACT: Deregulated Ras signalling initiates and maintains melanocyte neoplasia. The Rho-like GTPase Rac has been implicated in Ras-induced neoplastic transformation. Moreover, a recurrent UV-induced mutation activating RAC1 has recently been detected in human melanoma. Here, a role for Rac in melanoma initiation and progression was investigated in human melanomas and zebrafish models of melanocyte neoplasia. Immunohistochemical analysis revealed RAC expression and activity restricted to melanocytes at the junction of the epidermis and dermis in benign neoplasms. Malignant melanocytes displayed elevated RAC activity that extended into the suprabasal epidermis, deeper into the dermis, and was maintained in metastases. Previously, we have used zebrafish transgenic models to demonstrate that deregulated Ras-Raf-Mapk signalling can initiate melanocyte neoplasia. Expression of a constitutively active RAC1 mutant (V12RAC1) was not sufficient to initiate melanocyte neoplasia in this organism. Further, we did not detect an additive effect when combined with V600EBRAF; nor could V12RAC1 substitute for suppressed Pi3k signalling to restore melanoma progression. However, co-expression of V12RAC1 and oncogenic RAS accelerated tumour nodule formation. Immunohistochemical analysis revealed that the Rac activator Tiam1 is overexpressed in melanoma tumour nodules both in zebrafish and man. Thus our data suggest that Rac contributes to the progression of melanoma and that Tiam1 may activate Rac in nodular presentations.Journal of Investigative Dermatology accepted article preview online, 21 January 2013; doi:10.1038/jid.2013.23.
    Journal of Investigative Dermatology 01/2013;
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    ABSTRACT: Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis. Copyright © 2012 John Wiley & Sons, Ltd.
    Rapid Communications in Mass Spectrometry 01/2013; 27(1):127-34.
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    ABSTRACT: The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast , whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the and genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.
    PLoS ONE 01/2013; 8(2):e57846.
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    ABSTRACT: This chapter reviews the current treatment of chronic and neoplastic human papillomavirus (HPV)-associated conditions and the development of novel therapeutic approaches. Surgical excision of HPV-associated lower genital tract neoplasia is very successful but largely depends on secondary prevention programmes for identification of disease. Only high-risk HPV-driven chronic, pre-neoplastic lesions and some very early cancers cannot be successfully treated by surgical procedures alone. Chemoradiation therapy of cervical cancer contributes to the 66-79% cervical cancer survival at 5 years. Outlook for those patients with persistent or recurrent cervical cancer following treatment is very poor. Topical agents such as imiquimod (immune response modifier), cidofovir (inhibition of viral replication; induction apoptosis) or photodynamic therapy (direct damage of tumour and augmentation of anti-tumour immunity) have all shown some useful efficacy (∼50-60%) in treatment of high grade vulvar intraepithelial neoplasia (VIN). Provider administered treatments of genital warts include cryotherapy, trichloracetic acid, or surgical removal which has the highest primary clearance rate. Patient applied therapies include podophyllotoxin and imiquimod. Recurrence after "successful" treatment is 30-40%. Further improvements could derive from a rational combination of current therapy with new drugs targeting molecular pathways mediated by HPV in cancer. Small molecule inhibitors targeting the DNA binding activities of HPV E1/E2 or the anti-apoptotic consequences of E6/E7 oncogenes are in preclinical development. Proteasome and histone deacetylase inhibitors, which can enhance apoptosis in HPV positive tumour cells, are being tested in early clinical trials. Chronic high-risk HPV infection/neoplasia is characterised by systemic and/or local immune suppressive regulatory or escape factors. Recently two E6/E7 vaccines have shown some clinical efficacy in high grade VIN patients and this correlated with strong and broad systemic HPV-specific T cell response and modulation of key local immune factors. Treatments that can shift the balance of immune effectors locally in combination with vaccination are now being tested. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.
    Vaccine 11/2012; 30 Suppl 5:F71-82.
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    ABSTRACT: PURPOSE: Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are predicted to be responsible for tumour initiation, maintenance and metastases. Interleukin-8 (IL-8) is upregulated in breast cancer and associated with poor prognosis. Breast cancer cell line studies indicate that IL-8 via its cognate receptors, CXCR1 and CXCR2, is important in regulating breast CSC activity. We investigated the role of IL-8 in the regulation of CSC activity using patient-derived breast cancers and determined the potential benefit of combining CXCR1/2 inhibition with HER2-targeted therapy. Experimental design: CSC activity of metastatic and invasive human breast cancers (n=19) was assessed ex vivo using the mammosphere colony forming assay. RESULTS: Metastatic fluid IL-8 level correlated directly with mammosphere formation (r=0.652; P<0.05; n=10). Recombinant IL-8 directly increased mammosphere formation/self-renewal in metastatic and invasive breast cancers (n=17). IL-8 induced activation of EGFR/HER2 and downstream signalling pathways and effects were abrogated by inhibition of SRC, EGFR/HER2, PI3K or MEK. Furthermore, lapatinib inhibited the mammosphere-promoting effect of IL-8 in both HER2-positive and negative patient-derived cancers. CXCR1/2 inhibition also blocked the effect of IL-8 on mammosphere formation and added to the efficacy of lapatinib in HER2-positive cancers. CONCLUSIONS: These studies establish a role for IL-8 in the regulation of patient-derived breast CSC activity and demonstrate that IL-8/CXCR1/2 signalling is partly mediated via a novel SRC and EGFR/HER2-dependent pathway. Combining CXCR1/2 inhibitors with current HER2-targeted therapies has potential as an effective therapeutic strategy to reduce CSC activity in breast cancer and improve the survival of HER2-positive patients.
    Clinical Cancer Research 11/2012;
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    ABSTRACT: Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin-dependent kinase (CDK) and the onset of septation, before G1-CDK can be reactivated and drive re-polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1-CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN-dependent release of Cdc14, which counteracts all classes of CDK-mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis.
    The EMBO Journal 08/2012; 31(17):3620-34.
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