A proteomic analysis of Raf-1 signalling pathways

Raf-1 is an integral part of the Ras/Raf/MEK/ERK signalling pathway that relays mitogenic and survival signals to the nucleus, leading to suppression of apoptosis, growth and proliferation. The ability of Raf-1 to fulfil these diverse functions is likely to require its ability to form multiple complexes which are regulated by phosphorylation and sub-cellular localisation. The aim of the work described in this thesis was to develop proteomic approaches to study dynamic changes in Raf-1 signalling complexes in vivo. Recent studies have indicated that Raf-1 not only plays a crucial role in relaying growth signals through the MAPK pathway but it also has a kinase independent function in protecting cells from apoptosis. To characterise this anti-apoptotic role of Raf-1, DIGE (different gel electrophoresis) was used to determine changes in protein expression resulting from the reconstitution of Raf-1-/- fibroblasts with kinase-dead or wild-type Raf-1. Numerous pro- and anti-apoptotic proteins were differentially expressed between these three cell lines. Furthermore levels of several potential Raf-1 binding proteins and proteins involved in actin dynamics and motility were altered. The changes in protein expression identified by proteomic analysis are consistent with the hypersensitivity of Raf-l-/- fibroblasts to apoptotic stimuli and the alterations in their actin cytoskeleton and motility. In addition to examining global protein changes by DIGE, we developed a gel-less, mass spectrometry based two-dimensional liquid chromatography approach to analyse protein samples of medium complexity and successfully adapted it to identify Raf-1 protein complexes. We identified over 100 potential Raf-1 interacting partners, some of which required the presence of S259 site to bind to Raf-1 whereas others interacted only when cells were induced with mitogens or apoptotic insults. Two of these interactions (protein phosphatase 5 and SIP1) were verified by co-immunoprecipitations and functional analysis was performed on one of these proteins. Furthermore we developed a highly sensitive and robust method for phosphorylation site mapping and demonstrated its potential by identifying phosphorylation sites of proteins present in Raf-1 complexes. Raf-1 activation is a complex process in which phosphorylation plays a key role. While several kinases have been shown to phosphorylate Raf-1, only one phosphatase, PP2A, has been identified to regulate Raf-1 activity in vivo by dephosphorylating serine 259 upon activation with mitogens. We identified protein phosphatase 5 (PP5) as a potential Raf-1 interacting protein. This interaction was validated by co-immunoprecipitation of endogenous Raf-1 with PP5 in COS-1 cells. PP5 dissociated from Raf-1 upon stimulation with EGF and specifically dephosphorylated a crucial activation site, S338, in-vivo and in- vitro. Furthermore we provide evidence that cross talk between the MAPK pathway and the Gal2 pathway is regulated by PP5. The results presented here show that we have developed robust proteomic methods enabling us to analyse protein complexes as well as changes in protein expression and phosphorylation with high sensitivity and accuracy

Strongly truncated Dnaaf4 plays a conserved role in Drosophila ciliary dynein assembly as part of an R2TP-like co-chaperone complex with Dnaaf6

Axonemal dynein motors are large multi-subunit complexes that drive ciliary movement. Cytoplasmic assembly of these motor complexes involves several co-chaperones, some of which are related to the R2TP co-chaperone complex. Mutations of these genes in humans cause the motile ciliopathy, Primary Ciliary Dyskinesia (PCD), but their different roles are not completely known. Two such dynein (axonemal) assembly factors (DNAAFs) that are thought to function together in an R2TP-like complex are DNAAF4 (DYX1C1) and DNAAF6 (PIH1D3). Here we investigate the Drosophila homologues, CG14921/Dnaaf4 and CG5048/Dnaaf6. Surprisingly, Drosophila Dnaaf4 is truncated such that it completely lacks a TPR domain, which in human DNAAF4 is likely required to recruit HSP90. Despite this, we provide evidence that Drosophila Dnaaf4 and Dnaaf6 proteins can associate in an R2TP-like complex that has a conserved role in dynein assembly. Both are specifically expressed and required during the development of the two Drosophila cell types with motile cilia: mechanosensory chordotonal neurons and sperm. Flies that lack Dnaaf4 or Dnaaf6 genes are viable but with impaired chordotonal neuron function and lack motile sperm. We provide molecular evidence that Dnaaf4 and Dnaaf6 are required for assembly of outer dynein arms (ODAs) and a subset of inner dynein arms (IDAs)

Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib

RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.European Commission Horizon 2020Science Foundation Irelan

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses

We show theoretically and experimentally a mechanismbehind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input-output characteristics (the dose-response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose-response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose-response obtained experimentally

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosoliccomponents. The final degradation step is essential for clearing autophagic cargo and recyclingmacromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conservedtransmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation

Hypoxia Reduces the Pathogenicity of Pseudomonas Aeruginosa by Decreasing the Expression of Multiple Virulence Factors

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases

Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

Background & AimsIn the developing liver, bipotent epithelial progenitor cells known as hepatoblasts undergo lineage segregation to form the two major epithelial cell types, hepatocytes that constitute the bulk of the liver parenchyma and biliary epithelial cells (cholangiocytes) which comprise the bile duct, a complex tubular network which is critical for normal liver function. Notch and TGFβ signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from simple sheet of epithelial cells to a complex and connected bile duct.MethodsBy combining single cell RNA sequencing from embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning.ResultsWe show that the planar cell polarity protein, VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2-function results in the abnormal distribution of cortical actin remodelling resulting in the failure of bile duct formation.ConclusionsPlanar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture.Impact and ImplicationsHuman disease and mouse models have allowed us to define how the mammalian biliary lineage is specified during liver development. Once this relatively simple epithelium has formed though, how it undergoes morphogenesis to form a complex and branched structure is not clear. Similar to other branched tissues such as the liver and kidney the bile ducts use planar cell polarity signalling to coordinate cell movements; however how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein, VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are pattered across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

Objective: Immunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack of response. Here, we aimed to further investigate the immunoregulatory function of focal adhesion kinase (FAK) in PDAC, with specific emphasis on regulation of the type-II interferon response that is critical in promoting T-cell tumour recognition and effective immunosurveillance. Design: We combined CRISPR, proteogenomics and transcriptomics with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and validated findings using proteomic analysis of human patient-derived PDAC cell lines and analysis of publicly available human PDAC transcriptomics datasets. Results: Loss of PDAC cell-intrinsic FAK signalling promotes expression of the immunoproteasome and Major Histocompatibility Complex class-I (MHC-I), resulting in increased antigen diversity and antigen presentation by FAK-/- PDAC cells. Regulation of the immunoproteasome by FAK is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high affinity binding to MHC-I. Expression of these pathways can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, resulting in extensive infiltration of tumour-reactive CD8 T-cells and further restraint of tumour growth. FAK-dependent regulation of antigen processing and presentation is conserved between mouse and human PDAC, but is lost in cells/tumours with an extreme squamous phenotype. Conclusion: Therapies aimed at FAK degradation may unlock additional therapeutic benefit for the treatment of PDAC through increasing antigen diversity and promoting antigen presentation