The RHO Family GTPases: Mechanisms of Regulation and Signaling

Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed

Quantification of Triple Single-Leg Hop Test Temporospatial Parameters: A Validated Method Using Body-Worn Sensors for Functional Evaluation after Knee Injury

Lower extremity kinematic alterations associated with sport-related knee injuries may contribute to an unsuccessful return to sport or early-onset post-traumatic osteoarthritis. Also, without access to sophisticated motion-capture systems, temporospatial monitoring of horizontal hop tests during clinical assessments is limited. By applying an alternative measurement system of two inertial measurement units (IMUs) per limb, we obtained and validated flying/landing times and hop distances of triple single-leg hop (TSLH) test against motion-capture cameras, assessed these temporospatial parameters amongst injured and uninjured groups, and investigated their association with the Knee Injury and Osteoarthritis Outcome Score (KOOS). Using kinematic features of IMU recordings, strap-down integration, and velocity correction techniques, temporospatial parameters were validated for 10 able-bodied participants and compared between 22 youth with sport-related knee injuries and 10 uninjured youth. With median (interquartile range) errors less than 10(16) ms for flying/landing times, and less than 4.4(5.6)% and 2.4(3.0)% of reference values for individual hops and total TSLH progression, differences between hopping biomechanics of study groups were highlighted. For injured participants, second flying time and all hop distances demonstrated moderate to strong correlations with KOOS Symptom and Function in Daily Living scores. Detailed temporospatial monitoring of hop tests is feasible using the proposed IMUs system.Medicine, Faculty ofOther UBCNon UBCPhysical Therapy, Department ofReviewedFacult

RHO GTPase-Related Long Noncoding RNAs in Human Cancers

RHO GTPases are critical signal transducers that regulate cell adhesion, polarity, and migration through multiple signaling pathways. While all these cellular processes are crucial for the maintenance of normal cell homeostasis, disturbances in RHO GTPase-associated signaling pathways contribute to different human diseases, including many malignancies. Several members of the RHO GTPase family are frequently upregulated in human tumors. Abnormal gene regulation confirms the pivotal role of lncRNAs as critical gene regulators, and thus, they could potentially act as oncogenes or tumor suppressors. lncRNAs most likely act as sponges for miRNAs, which are known to be dysregulated in various cancers. In this regard, the significant role of miRNAs targeting RHO GTPases supports the view that the aberrant expression of lncRNAs may reciprocally change the intensity of RHO GTPase-associated signaling pathways. In this review article, we summarize recent advances in lncRNA research, with a specific focus on their sponge effects on RHO GTPase-targeting miRNAs to crucially mediate gene expression in different cancer cell types and tissues. We will focus in particular on five members of the RHO GTPase family, including RHOA, RHOB, RHOC, RAC1, and CDC42, to illustrate the role of lncRNAs in cancer progression. A deeper understanding of the widespread dysregulation of lncRNAs is of fundamental importance for confirmation of their contribution to RHO GTPase-dependent carcinogenesis

Selectivity Determinants of RHO GTPase Binding to IQGAPs

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions

Novel de novo CDC42 variants cause variable functional alterations and heterogeneous spectrum of neurodevelopmental and immune-hematologic rare diseases

CDC42 (Cell Division Cycle 42) is a small GTPase of the RAS super family regulating key developmental processes. We have recently characterized different CDC42 missense mutations associated with a broad spectrum of immune-haematological and neurodevelopmental disorders, which include RASopathies-related phenotypes. Here, we report the identification and molecular characterization of six additional CDC42 variants (p.K16R, p.Y23H, p.P34L, p.D118G, p.G164R, and p.D170N) associated with neurodevelopmental and immune-haematological conditions. In particular, patients present with various degree of cardiac defects, neurodevelopmental delay, facial dysmorphisms, and recurrent infections. CDC42 mutants variably affect protein expression and localization (Fig. 1) , GTPase activity (Fig. 2A), effector binding (i.e. RHOGDI (Fig. 2B), IQGAP1 (Fig. 3A), N-WASP (Fig. 3B), and, and RAS-mitogen-activated protein kinase (MAPK) pathway (Fig. 4). The comparative functional analysis of the CDC42 variants so far described indicates that only the IQGAP1-binding defective CDC42 mutants are associated with the immune-hematologic phenotype, suggesting a common pathogenic mechanism for these variants. Moreover, the majority of CDC42 variants significantly increases MAPK activation, indicating a role of the RAS-MAPK pathway in the pathogenesis of CDC42-associated disorders. Finally, our study expands the spectrum of CDC42 pathogenic variants and confirms the relevance of functional validation of unclassified variants to assess their possible role in disease pathogenicity

Electrostatic forces mediate the specificity of RHO GTPase-GDI interactions

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylger-anylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We de-termined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain.

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a 'scaffolding domain' by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42

DataSheet1_New mechanistic insights into the RAS-SIN1 interaction at the membrane.pdf

Stress-activated MAP kinase-interacting protein 1 (SIN1) is a central member of the mTORC2 complex that contains an N-terminal domain (NTD), a conserved region in the middle (CRIM), a RAS-binding domain (RBD), and a pleckstrin homology domain. Recent studies provided valuable structural and functional insights into the interactions of SIN1 and the RAS-binding domain of RAS proteins. However, the mechanism for a reciprocal interaction of the RBD-PH tandem with RAS proteins and the membrane as an upstream event to spatiotemporal mTORC2 regulation is not clear. The biochemical assays in this study led to the following results: 1) all classical RAS paralogs, including HRAS, KRAS4A, KRAS4B, and NRAS, can bind to SIN1-RBD in biophysical and SIN1 full length (FL) in cell biology experiments; 2) the SIN1-PH domain modulates interactions with various types of membrane phosphoinositides and constantly maintains a pool of SIN1 at the membrane; and 3) a KRAS4A-dependent decrease in membrane binding of the SIN1-RBD-PH tandem was observed, suggesting for the first time a mechanistic influence of KRAS4A on SIN1 membrane association. Our study strengthens the current mechanistic understanding of SIN1-RAS interaction and suggests membrane interaction as a key event in the control of mTORC2-dependent and mTORC2-independent SIN1 function.</p