2,004,117 research outputs found
Transcription Factor AP-2 Regulatory Signatures in Breast Cancer
No full textPhDAP-2 transcription factors are highly conserved basic helix-span-helix proteins whose
members ((x, ß, y, S and c) are crucial regulators of bryonic development. They also
play an important role in human neoplasia. uohis ochemical studies have detected
high levels of AP-2y expression in primary tumo of breast cancer patients. This high
expression has been correlated with reduced survival in all patients and reduced survival
in an ERa positive subset treated with hormone therapy. In breast cancer cell lines, AP-
2 factors have been implicated in the regulation of the ERBB2 proto-oncogene and ERa.
In an effort to further understand the role of AP-2y in breast carcinoma, this study has
sought to identify additional AP-2 activated cellular pathways and ultimately novel
transcriptional targets for AP-2 through the use of gene expression profiling.
RNAi using three independent AP-2y targeting sequences, has been used to deplete AP-
2y levels in the ERa positive MCF-7 breast carcinoma cell line, chosen as it exclusively
expresses the AP-2y family member. Microarrays were then utilised to create an AP-2y
dependent transcription profile. Statistical comparisons between non-silencing control
siRNA and AP-2y targeting siRNA groups identified a total of 162 gene expression
changes (p<0.01). These changes implicate AP-2y in the control of cell cycle
progression and developmental signalling. Indeed a role for AP-2y in the control of cell
cycle, in particular at the GUS transition, has been verified using flow cytometry.
Several of these gene expression changes, including IGFBP3, Transgelin and
KIAA1324, have been confirmed using qPCR and immunoblotting.
Finally, elevated levels of p21 mRNA and protein have been observed following AP-2y
silencing in MCF-7 cells. Additionally, the activity of a p21 promoter reporter is
repressed following transfection with an AP-2y expression construct in HepG2 cells.
These results coupled with ChIP experiments showing AP-2y occupancy at the proximal
promoter region of p21 in cycling MCF-7 cells, implicate AP-2y in the repression of
p21 transcription and suggest a role for AP2y in- the, control of cell cycle in breast
carcinoma in part through the transcriptional repression of p21
Regulation of breast tumour cell survival by AP-2 transcription factors
No full textPhDAP-2 transcription factors are crucial regulators of embryonic development and also
play important roles in human neoplasia. Over-expression of AP-2α and AP-2γ in
primary breast cancer (BC) correlates with expression of two major breast markers,
ERBB2 and oestrogen receptor. High AP-2γ expression is associated with reduced
survival in BC patients, including those treated with hormone therapy. Our aim is to
define the pathways regulated by AP-2 factors in breast epithelial cells. Data from
AP-2γ depleted MCF-7 cells suggested a role in cell cycle control. Here, regulation
by AP-2α and AP-2γ in additional breast cancer cell lines with differing genetic
background is investigated. Cell cycle analysis of synchronized T47D cells, which
express both AP-2 isoforms but mutant p53, showed a reduction in G1 but increased
S and G2/M-phase populations when AP-2α and AP-2γ were silenced either
independently or together. Despite the lack of growth arrest, p21cip protein levels
increased following AP-2 silencing. ChIP analysis showed AP-2α and AP-2γ binding
at the p21cip/CDKN1A promoter. In addition, cyclin D3 protein levels increased
following AP-2 silencing and ChIP analysis showed AP-2α and AP-2γ binding to its
promoter. Luciferase reporter constructs carrying CCND3 promoter sequences were
repressed when co-transfected with AP-2α or AP-2γ expression constructs. These
findings demonstrate the importance of AP-2 factors in the control of cell cycle
regulation but illustrate cell-type differences in their mode of action. Further work
focused on MCF10A immortalised breast epithelial cells, which express both AP-2
isoforms and wild-type p53. AP-2 silenced MCF10A cells adopted a more rounded
phenotype suggestive of changes in cell adhesion which was also supported by
microarray analysis of their gene expression profile.
KIAA1324, a protein of unknown function with features of a membrane-bound
growth factor receptor, was found to be significantly down-regulated following AP-
2γ silencing in MCF-7 cells. Functional assays using i) inducible knock down of
KIAA1324 in MCF-7 cells, and ii) stable KIAA1324 overexpression in MCF10A
investigated if altering KIAA1324 expression level could affect cell growth.
KIAA1324 did not affect breast epithelial cell proliferation on plastic but may
contribute to the ability of MCF-7 cells to display anchorage-independent growth
A role for amphiphysin in AP-1/clathrin coat formation
Full text linkTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. Clathrin, in combination with different adaptor proteins, is the major coat protein for vesicle formation at the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized is the formation of clathrin coats for endocytosis at the plasma membrane involving the adaptor protein complex AP-2. Clathrin and AP-2 were shown to be at the centre of a complex interactome of proteins accessory to vesicle formation. Considerably less is known about the formation of clathrin coated carriers at the TGN and endosomes, where the adaptor protein complex AP-1 plays a major role.
In vitro studies showed the minimal requirements for association of AP-1 to liposomal membranes to be activated ARF1, phosphoinositides, and either sorting signals or unknown cytosolic factors. We have used a liposome floatation assay to identify cytosolic proteins collaborating with AP-1 at the membrane. Separation of proteins from bovine brain cytosol with several chromatographic methods yielded an active fraction containing amphiphysin 1, amphiphysin 2, and endophilin A1. All three proteins are expressed in brain and known to be involved in AP-2/clathrin coat formation. They consist of an N-terminal N-BAR (Bin, amphiphysin, Rvs) domain for dimerization and membrane binding and a C-terminal SH3 (Src homology 3) domain for interaction with dynamin and synaptojanin. Amphiphysin 1 and 2 in addition contain a middle domain with binding sites for adaptors and clathrin. It was proposed that amphiphysins and endophilin are targeted to membranes with high curvature, such as the neck of a forming vesicle, where they recruit dynamin and synaptojanin in preparation for vesicle fission and uncoating.
In this thesis, I bacterially expressed and purified all three proteins and tested them in the floatation assay for AP-1 membrane binding activity. Only amphiphysin 2 showed activity, both as a homodimer and as a heterodimer with amphiphysin 1. Activity depended on a motif that was shown to bind to AP-1, AP-2, and clathrin in GST pull-down experiments.
Endogenous amphiphysins in primary neurons, as well as transiently expressed in neuronal or fibroblast cell lines, co-localized with AP-1 at the TGN. In addition, when expressed at high levels in neuronal cells, amphiphysins aggregated and interfered dominantly with the TGN localization of AP-1. Both phenomena depended on the presence of the clathrin and adaptor interaction sequence in the amphiphysins. Furthermore, both amphiphysins could be cross-linked to AP-1 in vivo.
Our results indicate that amphiphysin 1 and 2 function not only in clathrin coated vesicle formation for endocytosis at the plasma membrane, but are also part of the machinery forming AP-1/clathrin coats at the TGN and endosomes. This suggests that the machineries for CCV formation with AP-1 and AP-2 at different locations in the cell share more components than previously anticipated
Recruitment of AP-1 clathrin adaptors to liposomal membranes
Full text linkProtein and membrane traffic between organelles within the endocytic and exocytic pathway is mediated most prominently by coated vesicles. These vesicles are formed by the assembly of cytosolic coat proteins onto the donor membrane, which deform it into a bud so that vesicles can pinch off. Clathrin with its associated adaptors, COPI and COPII are the three major coats. Various in vitro studies allowed insight into the mechanism of coat formation. COPI and COPII vesicle budding from chemically defined liposomes has been reconstituted in vitro, using pure coat compounds. Further, it has been demonstrated that cargo is sorted into these vesicles. The mechanism of clathrin-coated vesicle formation appears to be more complicated. The AP-1 clathrin adaptor is involved in vesicle formation at the transGolgi network and endosomes. This work presents an in vitro assay where AP-1 is recruited to peptidoliposomes, presenting covalently linked peptides corresponding to sorting signals. In this system, AP-1 recruitment depends on myristyolated ADP-ribosylation factor(ARF1), GTP or GMP-PNP, tyrosine signals and a small amount of phosphoinositides, most prominently phosphatidyl inositol 4,5bisphosphate. In such a minimal system AP-1 is recruited as a highmolecular weight complex indicating the formation of a precoat in the absence of clathrin. GTP hydrolysis, induced by ARF GTPase-activating protein(ARFGAP1), disassembled this complex. Further, AP-1 is able to enhance the GAP activity of ARFGAP1 on myristoylated ARF1, suggesting a regulatory function of GTP hydrolysis in early steps of coat recruitment. This work provides insights into the mechanism of AP-1 clathrin coat formation which might also be used to investigate the recruitment of other coats
Characterizing the role of p21-Activated Kinase 3 (PAK3) in AP-1-induced transformation
No full textIncludes bibliographical references.Previous studies identified p21-Activated Kinase 3 (PAK3), a serine/threonine kinase, as a potential AP-1 target gene. PAK3 has been implicated in a variety of pathological disorders and over-expression of other PAK-family members has been linked to cancer. In this study, we investigated AP-1 regulation of PAK3 expression and the role of PAK3 in cJun/AP-1-associated cellular transformation. Our results showed elevated PAK3 expression at both the mRNA and protein level in cJun-over-expressing Rat1a fibroblasts, as well as in transformed human fibroblasts. Elevated PAK3 protein levels were also seen in cervical, ovarian, oesophageal and breast cancer cells lines, while poor survival tracked with high PAK3 expression in ovarian cancer patient material. Elevated PAK3 levels appear to play no role in the proliferation of transformed or cancerous cells, however appears vital for the transformed morphology and actin distribution. These cytoskeletal changes seem to be the underlying force governing cellular migration, as inhibition of PAK3 significantly reduced the motility of both transformed fibroblasts and cancer cell lines. Our data shows that elevated PAK3 expression in response to AP-1 over-expression is regulated through the transcriptional activation of the PAK3 promoter by AP-1 binding directly to a single site in the promoter. We also show that constitutive activation of PAK3 results in changes in cJun phosphorylation and an increase in AP-1 activity, which can be inhibited by a serine/threonine kinase inhibitor. PAK3 and AP-1 proteins were also shown to directly interact with each other. Our study is a first to describe a role for AP-1 in regulating PAK3 expression, and PAK3 in regulating AP-1 activity, identifying a potential feedback loop in which PAK3 is an AP-1 target required for cytoskeletal reorganization and migration observed in transformed cells
A cytosolic factor mediating membrane recruitment of AP-1 clathrin adaptors
Full text linkTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. These vesicles are formed through the recruitment of cytosolic coat proteins to the donor membrane that act as a scaffold to form coated buds and vesicles. At the same time they selectively concentrate cargo proteins by interacting with cytosolic signals. Clathrin, in combination with different adaptor proteins (APs), is the major coat protein for vesicle formation at the plasma membrane, endosomes and the trans-Golgi network. Best characterized is clathrin mediated endocytosis at the plasma membrane which involves AP-2 and a network of associated proteins. Much less is known about AP-1 mediated clathrin coated vesicle formation at the TGN/endosomes. In vitro studies demonstrated that the minimal requirements to recruit AP-1 to liposome membranes are activated Arf1, phosphoinositides, and either sorting signals or an unknown cytosolic factor. In order to identify this factor, we fractionated calf brain cytosol by several chromatographic methods. Fractions were tested for factor dependent AP-1 recruitment activity using an in vitro assay. Purification via ammonium sulfate precipitation, hydrophobic interaction chromatography, anion/cation exchange chromatography or hydroxyapatite chromatography produced a final fraction containing three major proteins: amphiphysin 1, amphiphysinand endophilin A1. All three proteins are known accessory factors in clathrin coated vesicle formation at the plasma membrane. Co-immunodepletion of amphiphysinandresulted in a strong reduction of AP-1 recruitment activity. Therefore we conclude that a heterodimer of amphiphysinandis the long searched for cytosolic factor, required to recruit AP-1 in the absence of sorting signals in vitro. Our results strongly suggest that amphiphysin 1, amphiphysinand endophilin A1 are also involved in AP-1 mediated clathrin coated vesicle formation at the TGN and endosomes in vivo
WE TCP-AP: Wireless Enhanced TCP-AP
Full text linkCongestion control in wireless networks is strongly dependent on the dynamics and instability of wireless links. It is known that TCP experiences serious performance degradation problems in wireless networks. New approaches based on TCP try to overcome these problems but, although their performance is increased, they incur in congestion control errors, since they do not evaluate accurately the capacity and available link bandwidth in wireless networks. This is also the case of TCP-AP (Adaptive Pacing) that, although presenting clear advantages in wireless networks when compared to other TCP-based approaches, its performance is still lower than rate-based approaches. In this paper we propose a new congestion control protocol based in TCP-AP, the Wireless Enhanced TCP-AP (WE TCPAP).This protocol relies on the MAC layer information gathere by a new method to accurately estimate the available bandwidth and the path capacity over a wireless network path. The new congestion control mechanism is evaluated in different scenarios in wireless mesh and ad-hoc networks, and compared against several approaches for wireless congestion control. It is shown that the new WE TCP-AP outperforms the base TCP-AP, with a more stable behavior and better channel utilization, and its performance gets close to the one of ratebased protocols. This is a very important result, as we show that TCP-based approaches are still able to have good performance in wireless mesh and ad-hoc networks
ap--/python-seabreeze: v2.5.0
No full text<h2>What's Changed</h2>
<ul>
<li>fix build with cython 3.0 by @ap-- in https://github.com/ap--/python-seabreeze/pull/210</li>
<li>zenodo batch by @ap-- in https://github.com/ap--/python-seabreeze/pull/212</li>
<li>Drop Python 3.7 by @ap-- in https://github.com/ap--/python-seabreeze/pull/219</li>
<li>SR2 support by @ap-- in https://github.com/ap--/python-seabreeze/pull/220</li>
</ul>
<p><strong>Full Changelog</strong>: https://github.com/ap--/python-seabreeze/compare/v2.4.0...v2.5.0</p>
ap-GnRH stimulated IP1 accumulation in S2 cells transfected with ap-GnRHR-L, but not ap-GnRHR-S.
No full textData are expressed as fold stimulation over IP1 levels in the untreated cells. Proctolin was used as a positive control in ap-GnRHR-L or ap-GnRHR-S-transfected cells, and empty vector-transfected cells were used as a negative control. N = 3 assays run in triplicates.</p
ap--/python-seabreeze: v2.8.0
No full text<h2>What's Changed</h2>
<ul>
<li>Basic HR2 support by @gabrielbenedikt in https://github.com/ap--/python-seabreeze/pull/242</li>
<li>Support for HR4 by @MicheleCotrufo in https://github.com/ap--/python-seabreeze/pull/244</li>
</ul>
<h2>New Contributors</h2>
<ul>
<li>@gabrielbenedikt made their first contribution in https://github.com/ap--/python-seabreeze/pull/242</li>
<li>@MicheleCotrufo made their first contribution in https://github.com/ap--/python-seabreeze/pull/244</li>
</ul>
<p><strong>Full Changelog</strong>: https://github.com/ap--/python-seabreeze/compare/v2.7.0...v2.8.0</p>
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