- Biochemical and biophysical research communications
- Published over 2 years ago
Macroautophagy is a conserved degradative pathway and its deterioration is linked to disturbances in cellular proteostasis and multiple diseases. Here, we show that the RAB GTPase RAB18 modulates autophagy in primary human fibroblasts. The knockdown of RAB18 results in a decreased autophagic activity, while its overexpression enhances the degradative pathway. Importantly, this function of RAB18 is dependent on RAB3GAP1 and RAB3GAP2, which might act as RAB GEFs and stimulate the activity of the RAB GTPase. Moreover, the knockdown of RAB18 deteriorates proteostasis and results in the intracellular accumulation of ubiquitinated degradation-prone proteins. Thus, the RAB GTPase RAB18 is a positive modulator of autophagy and is relevant for the maintenance of cellular proteostasis.
Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR-difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the X-ray structures, is the same for Ran and Ras. A new X-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr39 side chain of Ran between the γ-phosphate and Gln69 prevents the optimal positioning of the attacking water molecule by the Gln69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr25 to the α-phosphate in Ran. By integration of X-ray structure analysis, experimental and theoretical IR spectroscopy the catalytic center of the X-ray structural models are further refined to sub-Ångstrom resolution, allowing an improved understanding of catalysis.
- Combinatorial chemistry & high throughput screening
- Published over 4 years ago
The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzymecoupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.
Directional sensing, a process in which cells convert an external chemical gradient into internal signaling events, is essential in chemotaxis. We have previously shown that a Rho GTPase, RacE, regulates gradient sensing inDictyosteliumcells. Here, using affinity purification and mass spectrometry, we identified a novel RacE-binding protein, GflB, which contains a Ras GEF domain and a Rho GAP domain. Using biochemical and gene knockout approaches, we showed that GflB balances the activation of Ras and Rho GTPases which enables cells to precisely orient signaling events toward higher concentrations of chemoattractants. Furthermore, we found that GflB is located at the leading edge of migrating cells and this localization is regulated by the actin cytoskeleton and phosphatidylserine. Our findings provide a new molecular mechanism that connects directional sensing and morphological polarization.
The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.
Autosomal dominant mutations that activate the leucine-rich repeat kinase-2 (LRRK2) cause inherited Parkinson’s disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved Thr/Ser residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen derived B Cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2 phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase inactive LRRK2[D2017A] knock-in MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knock-in mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A, S935A] knock-in MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended towards toxicity. We report here the characterization of a Cdc42 selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analogue was identified via structure activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition towards Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as non-competitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42 selective allosteric inhibitor and a related analogue, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29-VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.
Rag GTPases assemble into heterodimeric complexes consisting of RagA or RagB and RagC or RagD in higher eukaryotes, or Gtr1 and Gtr2 in yeast, to relay amino acid signals toward the growth-regulating target of rapamycin complex 1 (TORC1). The TORC1-stimulating state of Rag GTPase heterodimers, containing GTP- and GDP-loaded RagA/B/Gtr1 and RagC/D/Gtr2, respectively, is maintained in part by the FNIP-Folliculin RagC/D GAP complex in mammalian cells. Here, we report the existence of a similar Lst4-Lst7 complex in yeast that functions as a GAP for Gtr2 and that clusters at the vacuolar membrane in amino acid-starved cells. Refeeding of amino acids, such as glutamine, stimulated the Lst4-Lst7 complex to transiently bind and act on Gtr2, thereby entailing TORC1 activation and Lst4-Lst7 dispersal from the vacuolar membrane. Given the remarkable functional conservation of the RagC/D/Gtr2 GAP complexes, our findings could be relevant for understanding the glutamine addiction of mTORC1-dependent cancers.