Guanine Nucleotide Exchange Factor Protein Research Articles

Rab32 family proteins regulate autophagosomal components recycling.

In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).

Abstract 477: Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers

Abstract Background: KRAS is the most frequently mutated oncogene with high prevalence in non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and pancreatic cancers (PAC). FDA currently approved sotorasib provides breakthrough therapy for cancer patients with KRASG12C mutation, however there is still high unmet medical need for new agents that target a broader KRAS mutated tumors. A new opportunity emerges to develop a pan KRAS inhibitor by suppressing the upstream guanine nucleotide exchange factor (GEF) protein son of sevenless 1 (SOS1). SOS1 is a key activator of KRAS and facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state. Binding to its catalytic domain, small molecule SOS1 inhibitor prevents KRAS activation and suppresses cancer cell proliferation. Material and Methods: Regor’s unique Computer Accelerated Rational Design (CARD) technology platform was applied to identify potent and selective SOS1 inhibitors. Biochemical assays and cellular assays were utilized to drive the structure-activity relationship (SAR). In vitro and in vivo target engagement were confirmed. In vivo efficacy study data were generated using lung and pancreatic cancer xenograft mouse models with KRAS mutation. Results: In vitro, RGT-018 blocked the interaction of KRAS::SOS1 with high selectivity, and inhibited proliferation of a broad spectrum of mutant KRAS-driven cancer cells as a single agent. Robust anti-proliferation activity was observed when RGT-018 was combined with MEK, KRASG12C, EGFR or CDK2/4/6 inhibitors in vitro. Oral administration of RGT-018 inhibited tumor growth and suppressed KRAS signaling pathway activation in tumor xenografts. Furthermore, combination with MEK or KRASG12C inhibitors led to profound tumor regression. Conclusions: The pharmacological properties of RGT-018 represent an attractive drug candidate with oral bioavailability for combination with targeted agents to treat a broader patient population driven by mutant KRAS. Citation Format: Fei Xiao, Kailiang Wang, Xinjuan Wang, Huijuan Li, Zhilong Hu, Wei Huang, Xiaoming Ren, Teng Feng, Lili Yao, Jing Lin, Chunlai Li, Liufeng Mei, Zhuanzhuan Zhang, Yangyang Liu, Xi Chen, Xiaotian Zhu, Wenge Zhong, Zhi Xie. Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 477.

Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma.

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein‐protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co‐elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic—Diffuse B‐cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

Turning Platelets Off and On: Role of RhoGAPs and RhoGEFs in Platelet Activity.

Platelet cytoskeletal reorganisation is a critical component of platelet activation and thrombus formation in haemostasis. The Rho GTPases RhoA, Rac1 and Cdc42 are the primary drivers in the dynamic reorganisation process, leading to the development of filopodia and lamellipodia which dramatically increase platelet surface area upon activation. Rho GTPases cycle between their active (GTP-bound) and inactive (GDP-bound) states through tightly regulated processes, central to which are the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs catalyse the dissociation of GDP by inducing changes in the nucleotide binding site, facilitating GTP binding and activating Rho GTPases. By contrast, while all GTPases possess intrinsic hydrolysing activity, this reaction is extremely slow. Therefore, GAPs catalyse the hydrolysis of GTP to GDP, reverting Rho GTPases to their inactive state. Our current knowledge of these proteins is constantly being updated but there is considerably less known about the functionality of Rho GTPase specific GAPs and GEFs in platelets. In the present review, we discuss GAP and GEF proteins for Rho GTPases identified in platelets, their regulation, biological function and present a case for their further study in platelets.

Abstract LB117: Identification of novel SOS1-K-Ras disruptors for NSCLC and colon cancers with K-Ras mutation

Abstract KRAS along with other RAS genes represents the most prevalent oncogene in human cancers. Atypical Ras signaling has been identified in more than 30% of all human cancers with the most common being lung, colon, and pancreatic cancers. In particular, K-Ras has been identified as the most important Ras protein in cancer research, implicated in over 21% of human cancers. Current strategies drug only specific K-Ras mutants, leaving open an unmet need for new agents that can address a broader patient population. A new opportunity is emerging for the development of a therapeutic Pan Ras inhibitor by targeting the upstream guanine nucleotide exchange factor (GEF) protein SOS (Son of sevenless). Disruption of SOS1- K-Ras mutant interaction is a good strategy to inhibit activation of mutant K-Ras. SOS1 inhibition through the protein-protein interaction (PPI) disruption assists in keeping K-Ras in the GDP inactive, rather than its active GTP-bound state. Inhibition of SOS1 results in blockade of the RAS-MEK-ERK pathway and lowering proliferation, in both WT and all mutant Ras forms. Here we discuss our approach to target SOS1 to control aberrant Ras signaling in NSCLC and colon cancers. We report the identification of novel potent SOS1-K-Ras disruptors using the SOS1-K-Ras PPI TR-FRET assay. Multiple series have been identified, with a good SAR trend. We have identified low nanomolar potent compounds that translate to modulation of p-ERK in cell based assays in K-Ras mutant cell lines. These compounds exhibited good ADME properties like solubility and metabolic stability. Further profiling of these compounds is ongoing. Citation Format: Sanjita Sasmal, Anuradha Ramanathan, Venkatesham Boorgu. Identification of novel SOS1-K-Ras disruptors for NSCLC and colon cancers with K-Ras mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB117.

Abstract 1273: Discovery of a potent, selective, and orally bioavailable SOS1 inhibitor, RMC-023, an in vivo tool compound that blocks RAS activation via disruption of the RAS-SOS1 interaction

Abstract The guanine nucleotide exchange factor (GEF) protein SOS1 activates RAS by promoting its conversion from the GDP-bound RAS(OFF) state to the GTP-bound RAS(ON) state. SOS1 catalyzes or accelerates this nucleotide exchange reaction in response to upstream signals conveyed by a range of growth factor receptors. It acts by promoting the release of tightly bound GDP and thereby facilitating the binding of GTP, which is present at higher intracellular concentrations than GDP, to generate RAS(ON). SOS1 itself is activated by RAS through the binding of RAS(ON) to an allosteric site on the SOS1 protein, which leads to a positive feedback loop between SOS1 and RAS that increases the amplitude and duration of RAS signaling. As a result, there is considerable potential for amplification of RAS signals by SOS1. For this reason, and because SOS1 is a convergent node downstream of RTK signaling, SOS1 represents an attractive therapeutic target in RAS driven cancers. We have developed a collection of novel, proprietary small molecule inhibitors of SOS1. Here we describe the preclinical profile of a potent, selective, and orally bioavailable in vivo tool compound, RM-023, which disrupts the critical interaction between KRAS and SOS1. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, and thereby inhibit RAS pathway signaling and RAS-driven cancer cell growth in vitro. Oral administration of RM-023 produced a dose-dependent suppression of tumor RAS pathway activation in vivo and inhibited tumor growth in preclinical xenograft models of diverse RAS-addicted cancers at well-tolerated doses. Enhanced anti-tumor activity in RAS-addicted cancer models was observed when RM-023 was administered in combination with other RAS pathway inhibitors. We believe SOS1 inhibition represents an attractive companion for combination with RAS-directed inhibitors and may have unique utility in select RAS-addicted tumor types. Citation Format: Andreas Buckl, Elsa Quintana, Grace J. Lee, Nataliya Shifrin, Mengqi Zhong, Lindsay S. Garrenton, David C. Montgomery, Carlos Stahlhut, Frances Zhao, Dan M. Whalen, Severin K. Thompson, Arlyn Tambo-ong, Micah Gliedt, John E. Knox, James J. Cregg, Naing Aay, Jong Choi, Bao Nguyen, Atti Tripathi, Ruiping Zhao, Mae Saldajeno-Concar, Abby Marquez, Daphne Hsieh, Laura L. McDowell, Elena S. Koltun, Alun Bermingham, David Wildes, Mallika Singh, Zhengping Wang, Richard Hansen, Jan A. Smith, Adrian L. Gill. Discovery of a potent, selective, and orally bioavailable SOS1 inhibitor, RMC-023, an in vivo tool compound that blocks RAS activation via disruption of the RAS-SOS1 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1273.

Host-Parasite Interaction of Atlantic salmon (Salmo salar) and the Ectoparasite Neoparamoeba perurans in Amoebic Gill Disease.

Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction, which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two-component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.

Targeting the RhoGEF βPIX/COOL-1 in Glioblastoma: Proof of Concept Studies.

Simple SummaryGlioblastoma (GBM) is an incurable disease with a 14-month average life-expectancy following diagnosis, and clinical management has not improved in four decades. GBM mortality is due to rapid tumour growth and invasion into surrounding normal brain. Invasive cells make complete surgical removal of the tumour impossible, and result in disease relapse. Thus, it is imperative that any new treatment strategy takes these invading cells into consideration. Bevacizumab (Bev), which prevents the formation of new blood vessels, is an FDA approved therapy, but it has failed to increase overall survival in GBM and has even been shown to increase tumour invasion in some cases. Complementary anti-invasive therapies are therefore urgently required to enhance bevacizumab efficacy. We have identified βPIX/COOL-1, a RhoGEF protein which plays an important role in GBM cell invasion and angiogenesis and could be a useful target in this setting.Glioblastoma (GBM), a highly invasive and vascular malignancy is shown to rapidly develop resistance and evolve to a more invasive phenotype following bevacizumab (Bev) therapy. Rho Guanine Nucleotide Exchange Factor proteins (RhoGEFs) are mediators of key components in Bev resistance pathways, GBM and Bev-induced invasion. To identify GEFs with enhanced mRNA expression in the leading edge of GBM tumours, a cohort of GEFs was assessed using a clinical dataset. The GEF βPix/COOL-1 was identified, and the functional effect of gene depletion assessed using 3D-boyden chamber, proliferation, and colony formation assays in GBM cells. Anti-angiogenic effects were assessed in endothelial cells using tube formation and wound healing assays. In vivo effects of βPix/COOL-1-siRNA delivered via RGD-Nanoparticle in combination with Bev was studied in an invasive model of GBM. We found that siRNA-mediated knockdown of βPix/COOL-1 in vitro decreased cell invasion, proliferation and increased apoptosis in GBM cell lines. Moreover βPix/COOL-1 mediated endothelial cell migration in vitro. Mice treated with βPix/COOL-1 siRNA-loaded RGD-Nanoparticle and Bev demonstrated a trend towards improved median survival compared with Bev monotherapy. Our hypothesis generating study suggests that the RhoGEF βPix/COOL-1 may represent a target of vulnerability in GBM, in particular to improve Bev efficacy.

Abstract 2585: Tools for the discovery of KRAS pathway inhibitors

Abstract The small GTPase, KRAS, is a known oncogene that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated KRAS is locked in the activated GTP bound state and facilitates enhanced Ras signaling in cancer cells. While being a desirable target, absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. There has been a resurgence of interest in discovery of KRAS modulators after recent identification of unique binding pockets and successful inhibition of the KRAS G12C mutant by covalent chemical modifiers. With five molecules in clinical trials, many researchers are joining the efforts to find new inhibitors. Ras-mediated signaling commonly occurs through the receptor tyrosine kinase SOS-Ras pathway. To facilitate drug discovery activities targeting this pathway, we have produced the full spectrum of pathway proteins including kinases, wild type and mutated KRAS, and the upstream guanine nucleotide exchange factor protein SOS1. We have also developed and validated a number of biochemical and biophysical assays for these targets. This poster will summarize our most recent assay development efforts for the KRAS mutants including two different assays that monitor SOS1-mediated KRAS nucleotide exchange activities. Molecules that target SOS1 can be characterized using nucleotide exchange and KRAS::SOS1 protein-protein interaction assays. Binding of molecules to KRAS and SOS1 can also be assessed with biophysical methods. These tools and assays will significantly advance the drug discovery efforts of the industry. Citation Format: Ekaterina Kuznetsova, Lauren McCauley, Elaine Steinke-Neal, Timothy Sullivan, Louis Primavera, Haiching Ma. Tools for the discovery of KRAS pathway inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2585.

Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network.

The Rho guanine nucleotide exchange factor (RGNEF) protein encoded by the ARHGEF28 gene has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Biochemical and pathological studies have shown that RGNEF is a component of the hallmark neuronal cytoplasmic inclusions in ALS-affected neurons. Additionally, a heterozygous mutation in ARHGEF28 has been identified in a number of familial ALS (fALS) cases that may give rise to one of two truncated variants of the protein. Little is known about the normal biological function of RGNEF or how it contributes to ALS pathogenesis. To further explore RGNEF biology we have established and characterized a yeast model and characterized RGNEF expression in several mammalian cell lines. We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein. Additionally, through unbiased screening using the split-ubiquitin system, we have identified RGNEF-interacting proteins, including two ALS-associated proteins. Functional characterization of other RGNEF interactors identified in our screen suggest that RGNEF functions as a microtubule regulator. Our findings indicate that RGNEF misfolding and toxicity may cause impairment of the microtubule network and contribute to ALS pathogenesis.

Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection.

The cuticle is the outermost layer of plant aerial tissue that interacts with the environment and protects plants against water loss and various biotic and abiotic stresses. ADP ribosylation factor guanine nucleotide exchange factor proteins (ARF-GEFs) are key components of the vesicle trafficking system. Our study discovers that AtMIN7, an Arabidopsis ARF-GEF, is critical for cuticle formation and related leaf surface defense against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). Our transmission electron microscopy and scanning electron microscopy studies indicate that the atmin7 mutant leaves have a thinner cuticular layer, defective stomata structure, and impaired cuticle ledge of stomata compared to the leaves of wild type plants. GC–MS analysis further revealed that the amount of cutin monomers was significantly reduced in atmin7 mutant plants. Furthermore, the exogenous application of either of three plant hormones—salicylic acid, jasmonic acid, or abscisic acid—enhanced the cuticle formation in atmin7 mutant leaves and the related defense responses to the bacterial Pto infection. Thus, transport of cutin-related components by AtMIN7 may contribute to its impact on cuticle formation and related defense function.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Ancient complement and lineage-specific evolution of the Sec7 ARF GEF proteins in eukaryotes.

Guanine nucleotide exchange factors (GEFs) are the initiators of signaling by every regulatory GTPase, which in turn act to regulate a wide array of essential cellular processes. To date, each family of GTPases is activated by distinct families of GEFs. Bidirectional membrane trafficking is regulated by ADP-ribosylation factor (ARF) GTPases and the development throughout eukaryotic evolution of increasingly complex systems of such traffic required the acquisition of a functionally diverse cohort of ARF GEFs to control it. We performed phylogenetic analyses of ARF GEFs in eukaryotes, defined by the presence of the Sec7 domain, and found three subfamilies (BIG, GBF1, and cytohesins) to have been present in the ancestor of all eukaryotes. The four other subfamilies (EFA6/PSD, IQSEC7/BRAG, FBX8, and TBS) are opisthokont, holozoan, metazoan, and alveolate/haptophyte specific, respectively, and each is derived from cytohesins. We also identified a cytohesin-derived subfamily, termed ankyrin repeat-containing cytohesin, that independently evolved in amoebozoans and members of the SAR and haptophyte clades. Building on evolutionary data for the ARF family GTPases and their GTPase-­activating proteins allowed the generation of hypotheses about ARF GEF protein function(s) as well as a better understanding of the origins and evolution of cellular complexity in eukaryotes.

Rab7a and Mitophagosome Formation.

The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.

Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs

The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by “monomeric” PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA.

A pull-down procedure for the identification of unknown GEFs for small GTPases

ABSTRACTMembers of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

Coevolution of RAC Small GTPases and their Regulators GEF Proteins.

RAC proteins are small GTPases involved in important cellular processes in eukaryotes, and their deregulation may contribute to cancer. Activation of RAC proteins is regulated by DOCK and DBL protein families of guanine nucleotide exchange factors (GEFs). Although DOCK and DBL proteins act as GEFs on RAC proteins, DOCK and DBL family members are evolutionarily unrelated. To understand how DBL and DOCK families perform the same function on RAC proteins despite their unrelated primary structure, phylogenetic analyses of the RAC, DBL, and DOCK families were implemented, and interaction patterns that may suggest a coevolutionary process were searched. Interestingly, while RAC and DOCK proteins are very well conserved in humans and among eukaryotes, DBL proteins are highly divergent. Moreover, correlation analyses of the phylogenetic distances of RAC and GEF proteins and covariation analyses between residues in the interacting domains showed significant coevolution rates for both RAC–DOCK and RAC–DBL interactions.

Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking.

Dopamine (DA) homeostasis is essential for a variety of brain activities. Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for normal DA homeostasis. However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood. Here we show that the Rho-family guanine nucleotide exchange factor protein Vav2 is required for DAT cell surface expression and transporter activity modulated by glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor Ret. Mice deficient in either Vav2 or Ret displayed elevated DAT activity, which was accompanied by an increase in intracellular DA selectively in the nucleus accumbens. Vav2(-/-) mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response. Our data demonstrate that Vav2 is a determinant of DAT trafficking in vivo and contributes to the maintenance of DA homeostasis in limbic DA neuron terminals.