82 research outputs found
Inhibition of Autophagy Protease ATG4B as a Therapeutic Option for Pancreatic Ductal Adenocarcinoma
No full textPancreatic ductal adenocarcinoma is a horrific disease, with only ~7% of
patients surviving more than 5 years. Growing evidence shows that autophagy
inhibition could reduce tumour progression and synergise with
chemotherapeutics. Autophagy is a recycling system within cells, which is
hijacked by cancer to sustain aberrant proliferation. ATG4B is a cystine
protease involved in the autophagy process. Knockout or inhibition of ATG4B
causes a reduction in autophagy. The aim of this thesis is to validate ATG4B
as a drug target for pancreatic ductal adenocarcinoma and to identify novel
small molecules and PROTACs that inhibit ATG4B activity. In Chapter 3
pancreatic ductal adenocarcinoma cell lines and ATG4B knockout cell lines
are characterised. In Chapter 4 these cell lines are used in the development of
a pancreatic ductal adenocarcinoma 3D model, which shows that ATG4B
knockout can cause spheroids to disintegrate. This is further investigated in
Chapter 5, which shows that ATG4B knockout in PANC-1 causes changes in
motility, clonality, protein and mRNA expression. Interestingly, b-catenin
expression is highly significantly reduced in ATG4B knockout PANC-1 cells,
which cannot be rescued with the addition of ATG4B back into ATG4B
knockout (Chapter 6). Finally, in Chapter 7 compounds and PROTACs were
screened for ATG4B inhibition and degradation, respectively. Overall, this
thesis of work shows that ATG4B knockout can impact spheroid formation,
motility, clonality, protein and mRNA expression, accompanied by a reduction
in b-catenin expression. These findings could hold great therapeutic
advancements for patients with pancreatic ductal adenocarcinoma
Methods in Molecular Biology, Volume 716: Drug Design and Discovery: Methods and Protocols
No full text
On programmed ribosomal frameshifting: the alternative proteomes
No full textFrameshifting results from two main mechanisms: genomic insertions or deletions (indels) or programmed ribosomal frameshifting. Whereas indels can disrupt normal protein function, programmed ribosomal frameshifting can result in dual-coding genes, each of which can produce multiple functional products. Here, I summarize technical advances that have made it possible to identify programmed ribosomal frameshifting events in a systematic way. The results of these studies suggest that such frameshifting occurs in all genomes, and I will discuss methods that could help characterize the resulting alternative proteomes
Targeting Deubiquitinating Enzymes (DUBs) That Regulate Mitophagy via Direct or Indirect Interaction with Parkin
No full textThe quality control of mitochondria is critical for the survival of cells, and defects in the pathways required for this quality control can lead to severe disease. A key quality control mechanism in cells is mitophagy, which functions to remove damaged mitochondria under conditions of various stresses. Defective mitophagy can lead to a number of diseases including neurodegeneration. It has been proposed that an enhancement of mitophagy can improve cell survival, enhance neuronal function in neurodegeneration and extend health and lifespans. In this review, we highlight the role of deubiquitinating enzymes (DUBs) in the regulation of mitophagy. We summarise the current knowledge on DUBs that regulate mitophagy as drug targets and provide a list of small molecule inhibitors that are valuable tools for the further development of therapeutic strategies targeting the mitophagy pathway in neurodegeneration
GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
No full textHigh-content screening is a useful tool to understand complex cellular processes and to identify
genes, proteins or small molecule compounds that modulate such pathways. High-content assays
monitor the function of a protein or pathway by visualizing a change in an image-based readout,
such as a change in the localization of a reporter protein. Examples of this can be the translocation
of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane.
One protein that is known to undergo such translocation is the Growth Factor Receptor-bound
protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor
receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously
to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the
assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early
stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and
internalization (Antczak and Djaballah, 2016)
A New Age in Functional Genomics Using CRISPR/Cas9 in Arrayed Library Screening
No full textCRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening
Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice
No full textPMCID: PMC3473050This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase
No full textGrowing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity
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