Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

Molecular subversion of Cdc42 signalling in cancer.

Cdc42 is a member of the Rho family of small GTPases and a master regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. This small G protein and its regulators have been the subject of many years of fruitful investigation and the advent of functional genomics and proteomics has opened up new avenues of exploration including how it functions at specific locations in the cell. This has coincided with the introduction of new structural techniques with the ability to study small GTPases in the context of the membrane. The role of Cdc42 in cancer is well established but the molecular details of its action are still being uncovered. Here we review alterations found to Cdc42 itself and to key components of the signal transduction pathways it controls in cancer. Given the challenges encountered with targeting small G proteins directly therapeutically, it is arguably the regulators of Cdc42 and the effector signalling pathways downstream of the small G protein which will be the most tractable targets for therapeutic intervention. These will require interrogation in order to fully understand the global signalling contribution of Cdc42, unlock the potential for mapping new signalling axes and ultimately produce inhibitors of Cdc42 driven signalling.

Pancytopenia, Recurrent Infection, Poor Wound Healing, Heterotopia of the Brain Probably Associated with A Candidate Novel de Novo CDC42 Gene Defect: Expanding the Molecular and Phenotypic Spectrum.

CDC42 (cell division cycle protein 42) belongs to the Rho GTPase family that is known to control the signaling axis that regulates several cellular functions, including cell cycle progression, migration, and proliferation. However, the functional characterization of the CDC42 gene in mammalian physiology remains largely unclear. Here, we report the genetic and functional characterization of a non-consanguineous Saudi family with a single affected individual. Clinical examinations revealed poor wound healing, heterotopia of the brain, pancytopenia, and recurrent infections. Whole exome sequencing revealed a de novo missense variant (c.101C > A, p.Pro34Gln) in the CDC42 gene. The functional assays revealed a substantial reduction in the growth and motility of the patient cells as compared to the normal cells control. Homology three-dimensional (3-D) modeling of CDC42 revealed that the Pro34 is important for the proper protein secondary structure. In conclusion, we report a candidate disease-causing variant, which requires further confirmation for the etiology of CDC42 pathogenesis. This represents the first case from the Saudi population. The current study adds to the spectrum of mutations in the CDC42 gene that might help in genetic counseling and contributes to the CDC42-related genetic and functional characterization. However, further studies into the molecular mechanisms that are involved are needed in order to determine the role of the CDC42 gene associated with aberrant cell migration and immune response.

Repolarization of HSC attenuates HSCs failure in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a bone marrow failure (BMF) syndrome associated with an increased risk of myelodysplasia and leukemia. The molecular mechanisms of SDS are not fully understood. We report that primitive hematopoietic cells from SDS patients present with a reduced activity of the small RhoGTPase Cdc42 and concomitantly a reduced frequency of HSCs polar for polarity proteins. The level of apolarity of SDS HSCs correlated with the magnitude of HSC depletion in SDS patients. Importantly, exogenously provided Wnt5a or GDF11 that elevates the activity of Cdc42 restored polarity in SDS HSCs and increased the number of HSCs in SDS patient samples in surrogate ex vivo assays. Single cell level RNA-Seq analyses of SDS HSCs and daughter cells demonstrated that SDS HSC treated with GDF11 are transcriptionally more similar to control than to SDS HSCs. Treatment with GDF11 reverted pathways in SDS HSCs associated with rRNA processing and ribosome function, but also viral infection and immune function, p53-dependent DNA damage, spindle checkpoints, and metabolism, further implying a role of these pathways in HSC failure in SDS. Our data suggest that HSC failure in SDS is driven at least in part by low Cdc42 activity in SDS HSCs. Our data thus identify novel rationale approaches to attenuate HSCs failure in SDS.

A modified method for preparation of fluorescent MantGDP bound CDC42.

Small GTPase cycled between the GDP-bound inactive state and GTP-bound active state, catalyzed by guanine nucleotide exchange factors (GEFs). Guanine nucleotide exchange assay was a direct way to investigate the specificity, activity, and kinetics of GEFs. The N-methylanthraniloyl derivative of GDP (mantGDP), which was bound to small GTPase, served as a substitution for labeled small GTPase involved in bioluminescent, colorimetric, or radioactive methods due to its safety and sensitivity. In this study, we present an economical and efficient approach to prepare qualified mantGDP-bound CDC42, a member of the Rho GTPase family. In our protocol, with a Kd value of 0.048 µM, alkaline phosphatase hydrolysis of CDC42 increased mantGDP binding affinity to CDC42, allowing mant-nucleotide associating onto CDC42 more easily. Only 1.5-fold molar excess of mantGDP was required to prepare mantGDP-bound CDC42 without nonhydrolyzable GTP analog and high performance liquid chromatography. The mantGDP-bound CDC42 was verified to be efficient for measuring the guanine nucleotide exchange activity of VAV2.

Characterization of guanine nucleotide exchange activity of DH domain of human FGD2.

FGD2, a member of FGD family, contains a Dbl homology domain (DH) and two pleckstrin homology domains segregated by a FYVE domain. The DH domain has been deduced to be responsible for guanine nucleotide exchange of CDC42 to activate downstream factors. Our aim was to build a prokaryotic expression system for the DH domain and to examine its guanine nucleotide exchange activity toward CDC42 in vitro. A recombinant vector, which was successfully constructed based on pGEX-6P-1, was employed to express the DH domain of human FGD2 (FGD2-DH) in E. coli BL21 (DE3). Purified FGD2-DH behaved as a homogeneous monomer with an estimated molecular weight that corresponded to the theoretical molecular weight and was predicted to be an α-helix protein by circular dichroism spectroscopy. FGD2-DH displayed weak guanine nucleotide exchange activity in vitro and very weak interactions with CDC42 following glutaraldehyde cross-linking.

Conformational control of small GTPases by AMPylation.

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

A Computational Protocol for Regulating Protein Binding Reactions with a Light-Sensitive Protein Dimer.

Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.

Towards the application of Tc toxins as a universal protein translocation system.

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

The Intrinsic GDP/GTP Exchange Activities of Cdc42 and Rac1 Are Critical Determinants for Their Specific Effects on Mobilization of the Actin Filament System.

The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.

Monitoring RhoGDI Extraction of Lipid-Modified Rho GTPases from Membranes Using Click Chemistry.

The posttranslational lipid modification of Rho GTPases is important for their proper subcellular localization and signal transduction. Rho GTPases terminate in a CaaX motif, in which the cysteine residue is modified with either a farnesyl or geranylgeranyl isoprenoid. RhoGDI renders Rho GTPases soluble by masking their lipid moieties. We recently identified that the brain-specific splice variant of Cdc42 (bCdc42) containing a noncanonical CCaX motif harbors a dual prenyl-palmitoyl modification that prevents its binding to RhoGDI. This chapter describes a method to analyze RhoGDI extraction of Rho GTPases containing different lipid modifications from membranes using a liposome reconstitution assay and click chemistry.

Designed Mutations Alter the Binding Pathways of an Intrinsically Disordered Protein.

Many cellular functions, including signaling and regulation, are carried out by intrinsically disordered proteins (IDPs) binding to their targets. Experimental and computational studies have now significantly advanced our understanding of these binding processes. In particular, IDPs that become structured upon binding typically follow a dock-and-coalesce mechanism, whereby the docking of one IDP segment initiates the process, followed by on-target coalescence of remaining IDP segments. Multiple dock-and-coalesce pathways may exist, but one may dominate, by relying on electrostatic attraction and molecular flexibility for fast docking and fast coalescing, respectively. Here we critically test this mechanistic understanding by designing mutations that alter the dominant pathway. This achievement marks an important step toward precisely manipulating IDP functions.

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor.

The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42.

Inhibition of Cdc42-intersectin interaction by small molecule ZCL367 impedes cancer cell cycle progression, proliferation, migration, and tumor growth.

Cdc42 is a member of the Rho family of small GTPases that are at the crossroads of major oncogenic signaling pathways involved in both lung and prostate cancers. However, the therapeutic potential of Cdc42 regulation is still unclear due to the lack of pharmacological tools. Herein, we report that ZCL367 is a bona fide Cdc42 inhibitor that suppressed cancer development and ZCL278 can act as a partial Cdc42 agonist. In lung cancer cell lines with varying EGFR and Ras mutations as well as both androgen-independent and androgen-dependent prostate cancer cell lines, ZCL367 impeded cell cycle progression, reduced proliferation, and suppressed migration. ZCL367 decreased Cdc42-intersectin interactions and reduced Cdc42-mediated filopodia formation. ZCL367 showed increased potency and selectivity for Cdc42 when compared to Rac1 and RhoA. ZCL367 reduced A549 tumorigenesis in a xenograft mouse model. Altogether, ZCL367 is a selective Cdc42 inhibitor and an excellent candidate for lead compound optimization for further anticancer studies.

A Tuba/Cdc42/Par6A complex is required to ensure singularity in apical domain formation during enterocyte polarization.

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization.

Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region.

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2.

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.