Secretion in 3T3-L1 adipocytes

Adipocytes secrete a range of proteins, called adipokines, with important roles in a variety of biological processes such as metabolism, cardiovascular function and immunity. ACRP30 (also known as adiponectin) is an adipokine that displays anti-diabetic and anti-inflammatory properties and may prevent atherosclerosis. It is secreted exclusively by adipocytes and circulating levels of ACRP30 are reduced in obesity and diabetes. Although this protein has been widely studied, little is known about the mechanisms of secretion of ACRP30 in adipocytes. Here I have examined secretion of ACRP30 in 3T3-L1 adipocytes with the aim of understanding the secretory pathways used by ACRP30 as it traffics to the cell surface. Using intracellular localisation techniques 1 have shown that ACRP30 overlaps with transferrin receptor positive membranes, evidence that ACRP30 traffics via the transferrin- receptor positive endosomal system. This was supported by results showing that ablation of the transferrin-receptor positive endosomes strongly inhibited ACRP30 secretion, I have also investigated the effects of overexpressing mutant forms of the small GTPases Rabl1 and Arf6, Overexpression of Rabl 1 S25N significantly reduced basal and insulin- stimulated ACRP30 secretion and I have demonstrated that Arf6 plays a role in ACRP30 secretion. With the aim of identifying new adipokines, I have conducted a proteomic analysis of the proteins secreted by 3T3-L1 adipocytes. In this study, I identified 25 proteins that had not previously been identified as adipocyte-secreted. In functional studies, two of these proteins, orosomucoid and Nm23 could be shown to affect insulin-stimulated glucose uptake. In monocyte adhesion assays orosomucoid had anti-inflammatory effects

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles

Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination

Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUbD2). Here, we present a 4.1 Å cryo-EM structure of IUbD2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUbD2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub-DNA complex. The IUbD2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUbD2 complex

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA

The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double‐stranded DNA via promoting a large‐scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary “Arm” ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1‐UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post‐translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA

Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphological changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent anti-proliferative effects with greatest activity in hematological cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy

Newsprint media representations of the introduction of the HPV vaccination programme for cervical cancer prevention in the UK (2005-2008)

In September 2008, the human papillomavirus (HPV) immunisation programme was introduced in the UK for schoolgirls aged between 12 and 18 years of age. The vaccine shows high efficacy in preventing infection against HPV types 16 and 18 responsible for 70% of cervical cancer. However, to be most effective, the vaccine needs to be administered before exposure to the viruses and therefore, ideally, before young people become sexually active. The introduction of any new vaccine, and perhaps particularly one given to young teenage girls to prevent a sexually transmitted cancer-causing virus, has the potential to attract a great deal of media attention. This paper reports on content analysis of 344 articles published between January 2005 and December 2008 in 15 UK newspapers. It includes both manifest and latent analysis to examine newsprint media coverage of the introduction of the HPV vaccination programme and its role in HPV advocacy. We concluded that the newspapers were generally positive towards the new HPV vaccination and that over the 4 years period the newsworthiness of the HPV vaccination programme increased. In 2008 two events dominated coverage, firstly, the introduction of the HPV programme in September 2008 and secondly, in August 2008 the diagnosis on camera of cervical cancer given to Jade Goody, a 27 year old mother of two, who gained fame and notoriety in the UK through her participation in several reality television shows.There are two conclusions from this study. Firstly, the positive media coverage surrounding the introduction of the HPV vaccination programme is to be welcomed as it is likely to contribute towards influencing public perceptions about the acceptability and need for HPV vaccination. Secondly, the focus on prevalence rates of HPV infection among women and on women's sexual behaviours, in relation to HPV vaccination 'encouraging' promiscuity, is an unhelpful aspect of media coverage

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

The Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis

The Rab6-binding kinesin, Rab6-KIFL, was identified in a two-hybrid screen for proteins that interact with Rab6, a small GTPase involved in membrane traffic through the Golgi apparatus. We find that Rab6-KIFL accumulates in mitotic cells where it localizes to the midzone of the spindle during anaphase, and to the cleavage furrow and midbody during telophase. Overexpression of Rab6-KIFL causes a cell division defect resulting in cell death. Microinjection of antibodies to Rab6-KIFL results in the cells becoming binucleate after one cell cycle, and time-lapse microscopy reveals that this is due to a defect in cleavage furrow formation and thus cytokinesis. These data show that endogenous Rab6-KIFL functions in cell division during cleavage furrow formation and cytokinesis, in addition to its previously described role in membrane traffic

Stimuli-responsive aggregation of high molar mass poly(N,N-diethylacrylamide)-b-poly(4-acryloylmorpholine) in tetrahydrofuran

The self-assembly of block copolymers constitutes a timely research area in polymer science with implications for applications like sensing or drug-delivery. In here, we investigate the unprecedented aggregation behaviour of high molecular weight block copolymer poly(N,N-diethylacrylamide)-b-poly(4-acryloylmorpholine) (PDEA-b-PAM) (Mn>400 kg/mol) in organic solvent tetrahydrofuran (THF). To elucidate the aggregation, dynamic light scattering, cryo transmission electron microscopy and turbidimetry were employed. The aggregate formation was assigned to the unprecedented upper critical solution temperature behaviour of PAM in THF at elevated concentration (> 6 wt%) and high molar masses. Various future directions for this new thermo-responsive block copolymer are envisioned, e.g. in the areas of photonics or templating of inorganic structures

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles