Hedgehog: functions and mechanisms Varjosalo, Markku; Taipale, Jussi
Genes & development,
09/2008, Letnik:
22, Številka:
18
Journal Article
Recenzirano
Odprti dostop
The Hedgehog (Hh) family of proteins control cell growth, survival, and fate, and pattern almost every aspect of the vertebrate body plan. The use of a single morphogen for such a wide variety of ...functions is possible because cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time cells are exposed to Hh. The Hh gradient is shaped by several proteins that are specifically required for Hh processing, secretion, and transport through tissues. The mechanism of cellular response, in turn, incorporates multiple feedback loops that fine-tune the level of signal sensed by the responding cells. Germline mutations that subtly affect Hh pathway activity are associated with developmental disorders, whereas somatic mutations activating the pathway have been linked to multiple forms of human cancer. This review focuses broadly on our current understanding of Hh signaling, from mechanisms of action to cellular and developmental functions. In addition, we review the role of Hh in the pathogenesis of human disease and the possibilities for therapeutic intervention.
Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics ...studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.
Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and ...complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.
Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs ...through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.
Individual cells in multicellular organisms constantly explore their microenvironment, or niche, to obtain spatial information that is used to regulate cell behavior to maintain tissue integrity. The ...extracellular matrix (ECM) is an important source of such spatial information. Binding of the integrin family receptors to the ECM triggers formation of integrin adhesion complexes (IACs) that link the ECM network to cellular cytoskeleton via remarkably large multiprotein complexes collectively referred to as the integrin adhesome. Recent advances in proteomics have enabled researchers to study the IAC composition in detail. Various biochemical IAC isolation methods and culture conditions have been employed to study the composition and dynamics of integrin‐mediated adhesions mainly in fibroblasts and lymphoblasts. These studies have led to identification of daunting lists of potential IAC components. This review focuses on the current status of proteomics‐driven research seeking to understand integrin functions by comprehensive analysis of IAC components. These systems level approaches have revealed the complexity of biochemical and biomechanical signals that are processed at IACs and provide a novel insight into how these signals are conveyed to regulate cellular behavior.
Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor ...suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2
breast cancer cells likewise led to increased MST1/2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2
MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2
breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2-induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.
Rhegmatogenous retinal detachment (RRD) is an ophthalmic emergency, which usually requires prompt surgery to prevent further detachment and restore sensory function. Although several individual ...factors have been suggested, a systems level understanding of molecular pathomechanisms underlying this severe eye disorder is lacking. To address this gap in knowledge we performed the molecular level systems pathology analysis of the vitreous from 127 patients with RRD using state-of-the art quantitative mass spectrometry to identify the individual key proteins, as well as the biochemical pathways contributing to the development of the disease. RRD patients have specific vitreous proteome profiles compared to other diseases such as macular hole, pucker, or proliferative diabetic retinopathy eyes. Our data indicate that various mechanisms, including glycolysis, photoreceptor death, and Wnt and MAPK signaling, are activated during or after the RRD to promote retinal cell survival. In addition, platelet-mediated wound healing processes, cell adhesion molecules reorganization and apoptotic processes were detected during RRD progression or proliferative vitreoretinopathy formation. These findings improve the understanding of RRD pathogenesis, identify novel targets for treatment of this ophthalmic disease, and possibly affect the prognosis of eyes treated or operated upon due to RRD.
In this study, we investigated the significance of a conserved five-amino acid motif 'AELPR' in the C-terminal region of helper component-proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) ...infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVAΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection.
Oncogenic gene fusions are estimated to account for up-to 20% of cancer morbidity. Recently sequence-level studies have established oncofusions throughout all tissue types. However, the functional ...implications of the identified oncofusions have often not been investigated. In this study, identified oncofusions from a fusion detection approach (DEEPEST) were analyzed in detail. Of the 28,863 oncofusions, we found almost 30% are expected to produce functional proteins with features from both parent genes. Kinases and transcription factors were the main gene families of the protein producing fusions. Considering their role as initiators, actors, and termination points of cellular signaling pathways, we focused our in-depth analyses on them. Domain architecture of the fusions and their wild-type interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins. To understand overall oncofusion effects, we performed differential expression analysis using TCGA cancer project samples. Results indicated oncofusion-specific alterations in gene expression levels, and lower expression levels of components of key cellular pathways, in particular signal transduction and transcription regulation. The sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling, possibly via acquiring novel functions.
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