Guanine Nucleotide Exchange Factor Activity Research Articles

Abstract 5912: Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer

Abstract Oncogenic Kras signaling is a key therapeutic target in human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major player that transduces receptor tyrosine kinase signaling to Ras. In addition to recently devised oncogenic Kras targeting small molecule inhibitors, disrupting the catalytic interaction between Ras and SOS1 has emerged as an attractive therapeutic strategy in working synergistically with Kras inhibitors and overcoming resistance. Previous studies have demonstrated an active “feed forward” loop of active Kras binding to the allosteric site of SOS1 that further stimulates SOS1 catalytic activation of wild type H/N/Kras, which is required for oncogenic Kras driven tumorigenesis. Here we have developed a rational approach that combines virtual screening with experimental screening to identify small molecule inhibitors that target the allosteric site of SOS1 to suppress oncogenic Kras mediated SOS1-regulated wild-type Ras activities. Unlike SOS1 catalytic site inhibitors which can have significant toxicity due to their ability to inhibit normal Ras signaling function, the lead SOS1 allosteric inhibitors developed in our laboratory binds to the allosteric site of SOS1 and blocks active Ras interaction with SOS1: we found these inhibitors can competitively suppresses SOS1-Ras interaction at the SOS1 allosteric site and dose-dependently inhibits the feed-forward SOS1 GEF activity. Mutagenesis and structure-activity relationship studies mapped the site of action to the SOS1 allosteric site, and preliminarily defined the chemical moieties in the inhibitor essential for the activity. The inhibitor showed dose-dependent and selective efficacy in inhibiting WT Ras activity, downstream signaling activities, and associated cell proliferation in KRAS mutation cells but not KRAS wild-type cells. Compared to the SOS1 catalytic inhibitor, BI3406, our inhibitors showed better selectivity for KRAS mutant pancreatic cancer cells vs. WT KRAS cells. These studies establish a proof of principle for the rational design of small molecule inhibitors targeting an oncogenic Kras specific allosteric site of SOS1 and provide a novel therapeutic concept to target KRAS-driven tumors more effectively. Citation Format: Xin Duan, Chris R Evelyn, Jacek Biesiada, James F Johnson, William L. Seibel, Jaroslaw Meller, Yi Zheng. Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5912.

Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, MglA, which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GAP (GTPase activating protein) and a GEF (guanine nucleotide exchange factor) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by co-expressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB1 and β0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.

Sec7 regulatory domains scaffold autoinhibited and active conformations

The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here, we report the cryogenic electron microscopy structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface.

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A.

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

Human DOCK11 Deficiency Causes Defective Erythropoiesis and Systemic Inflammation

Erythropoiesis involves significant changes in the cells, which are mediated by the plasma membrane and the actin cytoskeleton. The composition of the actin cytoskeleton and the interaction between its components are dynamically modified during erythropoiesis, however the precise role that different actin regulators play during erythroid differentiation is poorly understood. The dedicator of cytokinesis (DOCK) family member DOCK11 regulates actin cytoskeleton dynamics via its guanine nucleotide exchange factor (GEF) activity, resulting in activation of the small Rho GTPase CDC42. CDC42 has been implicated in regulating the early stages of erythroid development, as well as the terminal maturation steps involving enucleation. However, the role of human DOCK11 in hematopoietic cell function and human disease had not been defined. We analyzed a cohort of four patients from four unrelated families presenting with recurrent infections, early-onset severe immune dysregulation, systemic inflammation, as well as normocytic anemia and anisocytosis of unknown origin. Using whole-exome sequencing, we identified rare, hemizygous germline mutations in DOCK11 in these patients. Two mutations - an early stop-gain mutation and a To study the role of DOCK11 during erythropoiesis, we generated a dock11-knockout zebrafish model. We found that the dock11-knockout zebrafish embryos recapitulated the anemia and aberrant erythrocyte morphology observed in human DOCK11 deficiency. The anemia was amenable to rescue with constitutively active CDC42, suggesting that DOCK11 regulates erythrocyte numbers in a CDC42-dependent manner. As a next step we modeled human erythroid differentiation in vitro using an erythroid liquid culture system starting from purified CD34+ cells. ShRNA-mediated knockdown of DOCK11 in CD34 + cells revealed impairment of cell growth and differentiation during erythroid development. In line with moderate erythroid hypoplasia observed in the bone marrow of one of the patients, these data suggest an erythroid-intrinsic role of DOCK11 during erythropoiesis. As the patients with germline DOCK11 defects also presented with recurrent infections and systemic inflammation, we further assessed the role of DOCK11 in immune cells. In line with its role in actin dynamics, we found that human DOCK11 regulates T-cell morphology and migration, suggesting that defects in DOCK11 might impact the T cells' capability to fight infections. We further uncovered that the immune dysregulation observed in the patients involved aberrant T-cell activation and cytokine production. Mechanistically, using cells from DOCK11-deficient patients and Dock11-knockout mice, we were able to show that Dock11 regulates cytokine production at the transcriptional level by modulating the nuclear translocation of the T-cell transcription factor NFATc1, known to regulate the production of several cytokines. Collectively, we identified germline loss-of-function mutations affecting the actin regulator DOCK11 in a previously unknown disorder associating anemia and systematic inflammation. This work earmarks the DOCK11-CDC42 axis as an attractive future target for the treatment of a broader range of immune and hematological diseases

Ca2+-induced release of IQSEC2/BRAG1 autoinhibition under physiological and pathological conditions.

IQSEC2 (aka BRAG1) is a guanine nucleotide exchange factor (GEF) highly enriched in synapses. As a top neurodevelopmental disorder risk gene, numerous mutations are identified in Iqsec2 in patients with intellectual disabilities accompanied by other developmental, neurological, and psychiatric symptoms, though with poorly understood underlying molecular mechanisms. The atomic structures of IQSECs, together with biochemical analysis, presented in this study reveal an autoinhibition and Ca2+-dependent allosteric activation mechanism for all IQSECs and rationalize how each identified Iqsec2 mutation can alter the structure and function of the enzyme. Transgenic mice modeling two pathogenic variants of Iqsec2 (R359C and Q801P), with one activating and the other inhibiting the GEF activity of the enzyme, recapitulate distinct clinical phenotypes in patients. Our study demonstrates that different mutations on one gene such as Iqsec2 can have distinct neurological phenotypes and accordingly will require different therapeutic strategies.

Pathogen-specific structural features of Candida albicans Ras1 activation complex: uncovering new antifungal drug targets.

An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

Regulatory sites in the Mon1–Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

RhoA/ROCK signalling activated by ARHGEF3 promotes muscle weakness via autophagy in dystrophic mdx mice.

Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown. Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine. Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P<0.05) and in mice (+25%, P<0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P<0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P<0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles. Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.

ACE2 Activation by Tripeptide IRW (Ile-Arg-Trp) Depends on the G Protein-Coupled Receptor 30 Signaling Cascade

This study aimed to understand how specific cell-bound receptors influence ACE2 activation by IRW. Our results showed that G protein-coupled receptor 30 (GPR30), a 7-transmembrane domain protein, was involved in IRW-mediated ACE2 increase. IRW treatment (50 μM) significantly increased the GPR30 pool levels (3.2 ± 0.5 folds) (p < 0.001). IRW treatment also boosted the consecutive GEF (guanine nucleotide exchange factor) activity (2.2 ± 0.2 folds) (p < 0.001), and GNB1 levels (2.0 ± 0.5 folds) (p < 0.05), associated with the functional subunits of G proteins, in cells. These results were translated in hypertensive animal studies as well (p < 0.05), indicated by an increase in the aortal levels of GPR30 (p < 0.01); further experiments showed an increase in downstream PIP3/PI3K/Akt pathway activation following IRW treatment. The blockade of GPR30 by an antagonist and siRNA in cells abolished the ACE2-activating ability of IRW, as shown by the depleted levels of ACE2 mRNA (p < 0.001), protein levels in whole cells and membrane, angiotensin (1-7) (p < 0.01), and ACE2 promoter HNF1α (p < 0.05). Finally, the GPR30 blockade in ACE2-overexpressing cells using the antagonist (p < 0.01) and siRNA (p < 0.05) significantly depleted the innate cellular pool of ACE2, thus confirming the relationship between the membrane-bound GPR30 and ACE2. Overall, these results showed that the vasodilatory peptide IRW could activate ACE2 via the membrane-bound receptor GPR30.

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

Abstract A025: Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3

Abstract The MAPK/ERK signaling pathway is one of the most heavily mutated in human cancers. In normal cells, the stimulation of receptor tyrosine kinases (e.g. EGFR) on the plasma membrane signals the GTP-loading and activation of RAS through a mechanism catalyzed by the guanine nucleotide exchange factor (GEF) Son of Sevenless 1 (SOS1). RAS activation, in turn, initiates the RAF-MEK-ERK kinase cascade which triggers many downstream signals that can affect gene transcription, protein translation, cell proliferation, and feedback regulation. Several factors regulate SOS1 GEF activity, including two autoinhibitory domains within its own multi-domain architecture. First, the N-terminal domains of SOS1 occlude the allosteric RAS-GTP binding site in the RAS exchanger motif—thereby precluding SOS1 GEF activity—and this inhibition is relieved upon membrane recruitment and PIP2 phospholipid binding. Second, the C-terminal Proline-Rich Region (PRR) of SOS1 also occludes allosteric RAS-GTP binding by an unknown mechanism and is relieved when the growth factor receptor-bound protein 2 (Grb2) binds to motifs in the PRR. While the independent functions of these domains and interactions to inhibition and activation of GEF activity of SOS1 are known, the molecular mechanisms are poorly understood. An additional negative feedback loop involves the phosphorylation of the SOS1 PRR at two sites by the ribosomal protein S6 kinase 1 (RSK1), a substrate of downstream ERK. These modifications create 14-3-3 binding sites on SOS1, where 14-3-3 binding downregulates GEF activity, possibly by obstructing Grb2 association with the SOS1 PRR and membrane localization of SOS1. This work reconstitutes the SOS1:Grb2 and the SOS1:14-3-3 protein complexes in vitro to study the molecular mechanisms underlying SOS1 activity regulation. Citation Format: Orlando E. Martinez, Jawahar Sudhamsu. Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A025.

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

BackgroundC3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood.MethodsWe combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors.ResultsCrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G.ConclusionOur study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.ALj2d886i7aGyUezdi_xRMVideo abstract

Positive feedback in Ras activation through modulation of SOS activation probability.

Ras is a membrane-bound small GTPase that links receptor signals, such as from epidermal growth factor receptor (EGFR) and T cell receptor (TCR), to downstream MAPK signaling. Son of Sevenless (SOS), a guanine-nucleotide exchange factor (GEF), is a primary Ras activator in both EGFR and TCR signaling pathways. SOS is a unique Ras GEF because it contains both catalytic and allosteric Ras binding sites. An important feature of Ras-MAPK signaling is an apparent binary—or switch-like—activation response. This behavior has been attributed to active RasGRP-driven positive feedback in SOS and corresponding kinetic bistability. More recent single-molecule studies of SOS have revealed a variety of previously unknown behaviors, including strong processivity on the membrane (an individual SOS molecule can activate hundreds of Ras molecules during a single membrane binding event) and extended delay times between membrane recruitment and initiation of GEF activity. These studies have also revealed that the catalytic rate of Ras activation by SOS is independent of the nucleotide state of Ras in the allosteric binding site on SOS, thus raising questions about the origins of such positive feedback. This study aims to elucidate a refined mechanism for the observed positive feedback in SOS activation of Ras, which includes these more recent discoveries. Experiments and stochastic modeling studies focus on how the timing of SOS autoinhibition release, rather than common mechanisms such as product-mediated recruitment or allosterically enhanced catalytic rate, may give rise to the observed behavior.

PPDPF Promotes the Development of Mutant KRAS-Driven Pancreatic Ductal Adenocarcinoma by Regulating the GEF Activity of SOS1.

The guanine nucleotide exchange factor (GEF) SOS1 catalyzes the exchange of GDP for GTP on RAS. However, regulation of the GEF activity remains elusive. Here, the authors report that PPDPF functions as an important regulator of SOS1. The expression of PPDPF is significantly increased in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and recurrence of PDAC patients. Overexpression of PPDPF promotes PDAC cell growth in vitro and in vivo, while PPDPF knockout exerts opposite effects. Pancreatic-specific deletion of PPDPF profoundly inhibits tumor development in KRASG12D -driven genetic mouse models of PDAC. PPDPF can bind GTP and transfer GTP to SOS1. Mutations of the GTP-binding sites severely impair the tumor-promoting effect of PPDPF. Consistently, mutations of the critical amino acids mediating SOS1-PPDPF interaction significantly impair the GEF activity of SOS1. Therefore, this study demonstrates a novel model of KRAS activation via PPDPF-SOS1 axis, and provides a promising therapeutic target for PDAC.

Central nervous system specific high molecular weight ALS2/alsin homophilic complex is enriched in mouse brain synaptosomes

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.

Fission Yeast Rho1p-GEFs: From Polarity and Cell Wall Synthesis to Genome Stability.

Rho1p is a membrane-associated protein that belongs to the Rho family of small GTPases. These proteins coordinate processes such as actin remodelling and polarised secretion to maintain the shape and homeostasis of yeast cells. In response to extracellular stimuli, Rho1p undergoes conformational switching between a guanosine triphosphate (GTP)-bound active state and a guanosine diphosphate (GDP)-bound inactive state. Cycling is improved with guanine nucleotide exchange factor (GEF) activity necessary to activate signalling and GTPase activating protein (GAP) activity required for subsequent signal depletion. This review focuses on fission yeast Rho1p GEFs, Rgf1p, Rgf2p, and Rgf3p that belong to the family of DH-PH domain-containing Dbl-related GEFs. They are multi-domain proteins that detect biological signals that induce or inhibit their catalytic activity over Rho1p. Each of them activates Rho1p in different places and times. Rgf1p acts preferentially during polarised growth. Rgf2p is required for sporulation, and Rgf3p plays an essential function in septum synthesis. In addition, we outline the noncanonical roles of Rho1p-GEFs in genomic instability.

Structural biology of DOCK-family guanine nucleotide exchange factors.

DOCK proteins are a family of multi-domain guanine nucleotide exchange factors (GEFs) that activate the RHO GTPases CDC42 and RAC1, thereby regulating several RHO GTPase-dependent cellular processes. DOCK proteins are characterized by the catalytic DHR2 domain (DOCKDHR2 ), and a phosphatidylinositol(3,4,5)P3 -binding DHR1 domain (DOCKDHR1 ) that targets DOCK proteins to plasma membranes. DOCK-family GEFs are divided into four subfamilies (A to D) differing in their specificities for CDC42 and RAC1, and the composition of accessory signalling domains. Additionally, the DOCK-A and DOCK-B subfamilies are constitutively associated with ELMO proteins that auto-inhibit DOCK GEF activity. We review structural studies that have provided mechanistic insights into DOCK-protein functions. These studies revealed how a conserved nucleotide sensor in DOCKDHR2 catalyses nucleotide exchange, the basis for how different DOCK proteins activate specifically CDC42 and RAC1, and sometimes both, and how up-stream regulators relieve the ELMO-mediated auto-inhibition. We conclude by presenting a model for full-length DOCK9 of the DOCK-D subfamily. The involvement of DOCK GEFs in a range of diseases highlights the importance of gaining structural insights into these proteins to better understand and specifically target them.

Translationally controlled tumor protein: the mediator promoting cancer invasion and migration and its potential clinical prospects.

Translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein localized in the cytoplasm and nucleus of eukaryotic cells. It is secreted through exosomes and its degradation is associated with the ubiquitin-proteasome system (UPS), heat shock protein 27 (Hsp27), and chaperone-mediated autophagy (CMA). Its structure contains three α‍-helices and eleven β‍-strands, and features a helical hairpin as its hallmark. TCTP shows a remarkable similarity to the methionine-R-sulfoxide reductase B (MsrB) and mammalian suppressor of Sec4 (Mss4/Dss4) protein families, which exerts guanine nucleotide exchange factor (GEF) activity on small guanosine triphosphatase (GTPase) proteins, suggesting that some functions of TCTP may at least depend on its GEF action. Indeed, TCTP exerts GEF activity on Ras homolog enriched in brain (Rheb) to boost the growth and proliferation of Drosophila cells. TCTP also enhances the expression of cell division control protein 42 homolog (Cdc42) to promote cancer cell invasion and migration. Moreover, TCTP regulates cytoskeleton organization by interacting with actin microfilament (MF) and microtubule (MT) proteins and inducing the epithelial-mesenchymal transition (EMT) process. In essence, TCTP promotes cancer cell movement. It is usually highly expressed in cancerous tissues and thus reduces patient survival; meanwhile, drugs can target TCTP to reduce this effect. In this review, we summarize the mechanisms of TCTP in promoting cancer invasion and migration, and describe the current inhibitory strategy to target TCTP in cancerous diseases.

Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284–1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.