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.

Inhibition of the RacGEF VAV3 by the small molecule IODVA1 impedes RAC signaling and overcomes resistance to tyrosine kinase inhibition in acute lymphoblastic leukemia.

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

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.

Activating MRAS mutations cause Noonan syndrome associated with hypertrophic cardiomyopathy.

The RASopathies are a group of genetic syndromes caused by upregulated RAS signaling. Noonan syndrome (NS), the most common entity among the RASopathies, is characterized mainly by short stature, cardiac anomalies and distinctive facial features. Mutations in multiple RAS-MAPK pathway-related genes have been associated with NS and related phenotypes. We describe two unrelated patients presenting with hypertrophic cardiomyopathy (HCM) and dysmorphic features suggestive of NS. One of them died in the neonatal period because of cardiac failure. Targeted sequencing revealed de novo MRAS variants, c.203C > T (p.Thr68Ile) and c.67G > C (p.Gly23Arg) as causative events. MRAS has only recently been related to NS based on the observation of two unrelated affected individuals with de novo variants involving the same codons here found mutated. Gly23 and Thr68 are highly conserved residues, and the corresponding codons are known hotspots for RASopathy-associated mutations in other RAS proteins. Functional analyses documented high level of activation of MRAS mutants due to impaired GTPase activity, which was associated with constitutive plasma membrane targeting, prolonged localization in non-raft microdomains, enhanced binding to PPP1CB and SHOC2 protein, and variably increased MAPK and PI3K-AKT activation. This report provides additional evidence that a narrow spectrum of activating mutations in MRAS represents another rare cause of NS, and that MRAS has to be counted among the RASopathy genes predisposing to HCM. Moreover, our findings further emphasize the relevance of the MRAS-SHOC2-PPP1CB axis in the control of MAPK signaling, and the contribution of both MAPK and PI3K-AKT pathways in MRAS functional upregulation.

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.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

Prevalence and antimicrobial resistance patterns of Salmonella isolated from poultry farms in southeastern United States.

Salmonella spp. are among the most common foodborne pathogens, and increase in the occurrence of antimicrobial drug-resistant Salmonella poses a severe risk to public health. The main objective of this study was to determine changes in Salmonella prevalence and their antimicrobial resistance on poultry farms following recommendations to changes in biosecurity practices. Four poultry farms were sampled by collecting cloacal swabs, drag swabs, and litter samples prior to recommended biosecurity changes (March-April) and post recommendations (October-November). Prevalence of Salmonella was 3 to 4% during pre-recommendations, while the prevalence was higher (P > 0.05), ranging from 5 to 14% during post recommendations. Higher Salmonella prevalence was observed for pre- and post-recommendation phases by sample type in cloacal and drag samples -5% for farm 1, drag swab -6% on farm 2, cloacal swab -6% for farm 3, and drag swab -17% on farm 4. The PCR confirmed Salmonella were serotyped and tested for antimicrobial resistance. Six serotypes of Salmonella were identified with S. Enteritidis (52%) being the most prevalent, followed by S. Berta (38%), S. Mbandaka (7%), S. Typhimurium (2%), S. Kentucky (0.4%), and S. Tennessee (0.4%). A total of 7% isolates exhibited resistance to at least one of the 8 antimicrobials. Higher resistance was observed for tetracycline, streptomycin, and nalidixic acid. A single isolate of S. Mbandaka exhibited multidrug resistance to tetracycline, amoxicillin/clavulanic acid, and ampicillin. Based on these prevalence results, it can be inferred that, irrespective of implementation of improved biosecurity practices, seasonal variation can cause changes in the prevalence of Salmonella on the farms. Resistance to clinically important antimicrobials used to treat salmonellosis is a concern to public health.

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.

σ(B) plays a limited role in the ability of Listeria monocytogenes strain F2365 to survive oxidative and acid stress and in its virulence characteristics.

Listeria monocytogenes strain F2365 was the first strain representative of serotype 4b (lineage I) to be sequenced in 2004, suggesting it could become the model organism for this serotype, which is associated with most human outbreaks of listeriosis worldwide to date. F2365 itself is an outbreak strain that was involved in the listeriosis outbreak associated with Mexican-style soft cheese in California in 1985. In this study, we show through phenotypic and transcriptomic analysis that L. monocytogenes strain F2365 has reduced ability to respond to acid and oxidative stress. F2365 has neither the σ(B)-dependent ability to survive acid or oxidative stress nor the σ(B)-dependent ability to infect Caco-2 epithelial cells in vitro or guinea pigs in vivo. More studies are needed to determine whether the atypical σ(B)-independent response to stress observed in F2365 is strain specific, serotype specific, or even lineage specific.

Listeria monocytogenes {sigma}B has a small core regulon and a conserved role in virulence but makes differential contributions to stress tolerance across a diverse collection of strains.

Listeria monocytogenes strains are classified in at least three distinct phylogenetic lineages. There are correlations between lineage classification and source of bacterial isolation; e.g., human clinical and food isolates usually are classified in either lineage I or II. However, human clinical isolates are overrepresented in lineage I, while food isolates are overrepresented in lineage II. sigma(B), a transcriptional regulator previously demonstrated to contribute to environmental stress responses and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential abilities for L. monocytogenes survival in various niches (e.g., food and human clinical niches). To determine if the contributions of sigma(B) to stress response and virulence differ across diverse L. monocytogenes strains, DeltasigB mutations were created in strains belonging to lineages I, II, IIIA, and IIIB. Paired parent and DeltasigB mutant strains were tested for survival under acid and oxidative stress conditions, Caco-2 cell invasion efficiency, and virulence using the guinea pig listeriosis infection model. Parent and DeltasigB mutant strain transcriptomes were compared using whole-genome expression microarrays. sigma(B) contributed to virulence in each strain. However, while sigma(B) contributed significantly to survival under acid and oxidative stress conditions and Caco-2 cell invasion in lineage I, II, and IIIB strains, the contributions of sigma(B) were not significant for these phenotypes in the lineage IIIA strain. A core set of 63 genes was positively regulated by sigma(B) in all four strains; different total numbers of genes were positively regulated by sigma(B) in the strains. Our results suggest that sigma(B) universally contributes to L. monocytogenes virulence but specific sigma(B)-regulated stress response phenotypes vary among strains.

Listeria monocytogenes F2365 carries several authentic mutations potentially leading to truncated gene products, including inlB, and demonstrates atypical phenotypic characteristics.

Searches of the genome annotation of Listeria monocytogenes F2365, an isolate from the 1985 listeriosis epidemic in California, showed that this strain carries 20 authentic mutations resulting in premature stop codons, including a nonsense mutation in inlB. Here we showed that L. monocytogenes F2365 demonstrates atypical virulence-associated characteristics, including significantly (P < 0.05) reduced invasion efficiency in Caco-2 cells as compared with a closely related lineage I serotype 4b strain as well as significantly (P < 0.05) greater variation in invasiveness when grown under different conditions compared with standard laboratory control and other lineage I serotype 4b strains. In addition, L. monocytogenes F2365 demonstrated distinct growth characteristics, including a significantly (P < 0.05) reduced exponential growth rate when compared with laboratory control and other lineage I serotype 4b outbreak-associated strains as well as a significantly (P < 0.05) longer lag phase duration time compared with another lineage I serotype 4b strain. Our results support that L. monocytogenes F2365 is characterized by genotypic and phenotypic properties that are atypical of other L. monocytogenes strains.