Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small ...molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the ...role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a ...GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.
The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human ...Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners.
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•Structure of human Atg13-Atg101 HORMA dimer refined at 1.6 Å resolution•Atg13 safety belt anchored, ordered, and consistent with stable heterodimer•WF finger is in a closed and inactive conformation•Hydrophobic pockets marked by benzamidines suggest function as interaction hub
The human ULK1-FIP200-Atg13-Atg101 complex initiates autophagy and is a promising target for aging, neurodegeneration, cancer, and infection. Qi et al. describe a structure of the HORMA dimer of human Atg13 and Atg101 that shows how they assemble and might coordinate binding of substrates and regulatory proteins.
The striatin-interacting phosphatase and kinase (STRIPAK) complex is a large, multisubunit protein phosphatase 2A (PP2A) assembly that integrates diverse cellular signals in the Hippo pathway to ...regulate cell proliferation and survival. The architecture and assembly mechanism of this critical complex are poorly understood. Using cryo-EM, we determine the structure of the human STRIPAK core comprising PP2AA, PP2AC, STRN3, STRIP1, and MOB4 at 3.2-Å resolution. Unlike the canonical trimeric PP2A holoenzyme, STRIPAK contains four copies of STRN3 and one copy of each the PP2AA-C heterodimer, STRIP1, and MOB4. The STRN3 coiled-coil domains form an elongated homotetrameric scaffold that links the complex together. An inositol hexakisphosphate (IP
) is identified as a structural cofactor of STRIP1. Mutations of key residues at subunit interfaces disrupt the integrity of STRIPAK, causing aberrant Hippo pathway activation. Thus, STRIPAK is established as a noncanonical PP2A complex with four copies of regulatory STRN3 for enhanced signal integration.
RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding ...domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is
end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.
Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 ...subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
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•An essential autophagy factor Atg13 has a large intrinsically disordered region (IDR)•The IDR of Atg13 contains two distinct regions binding intermolecularly to Atg17•Tethering two Atg17 molecules by Atg13 leads to supramolecular Atg1 complex assembly•The Atg13-mediated supramolecular assembly is responsible for autophagy initiation
Yamamoto et al. uncover insights into early steps of yeast autophagosome assembly, where autophagy-related (Atg) proteins localize to a perivacuolar site to organize the pre-autophagosomal structure (PAS). Atg13, via an intrinsically disordered region, provides a flexible linkage between two Atg17 molecules to mediate multimeric Atg1 complex self-assembly for PAS organization.
At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of ...Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.
Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane
. Proteins with ...highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)
and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally
. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62-Sec63 complex
, and substrates are moved through the channel by the luminal BiP ATPase
. How the Sec62-Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70
. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.
Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses
. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene ...that is associated with systemic lupus erythematosus
-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease
. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF
. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus
.
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