Nucleotide Exchange Factor Domain Research Articles

Identification and characterization of human KALRN mRNA and Kalirin protein isoforms.

Kalirin is a multidomain protein with important roles in neurite outgrowth, and synaptic spine formation and remodeling. Genetic and pathophysiological links with various neuropsychiatric disorders associated with synaptic dysfunction and cognitive impairment have sparked interest in its potential as a pharmacological target. Multiple Kalirin proteoforms are detected in the adult human brain, yet we know little about the diversity of the transcripts that encode them or their tissue profiles. Here, we characterized full-length KALRN transcripts expressed in the adult human frontal lobe and hippocampus using rapid amplification of complementary DNA (cDNA) ends and nanopore long-read sequencing. For comparison with non-neural tissue, we also analyzed KALRN transcripts in the aorta. Multiple novel isoforms were identified and were largely similar between the two brain regions analyzed. Alternative splicing in the brain results in preferential inclusion of exon 37, which encodes 32 amino acids upstream of the second guanine nucleotide exchange factor (GEF) domain. Structural modeling predicts that a subset of these amino acids forms a conserved alpha helix. Although deletion of these amino acids had little effect on GEF activity, it did alter Kalirin-induced neurite outgrowth suggesting that this brain-enriched splicing event may be important for neural function. These data indicate that alternative splicing is potentially important for regulating Kalirin actions in the human brain.

Heterozygosity for neurodevelopmental disorder-associated TRIO variants yields distinct deficits in behavior, neuronal development, and synaptic transmission in mice.

Genetic variants in TRIO are associated with neurodevelopmental disorders (NDDs) including schizophrenia (SCZ), autism spectrum disorder (ASD) and intellectual disability. TRIO uses its two guanine nucleotide exchange factor (GEF) domains to activate GTPases (GEF1: Rac1 and RhoG; GEF2: RhoA) that control neuronal development and connectivity. It remains unclear how discrete TRIO variants differentially impact these neurodevelopmental events. Here, we investigate how heterozygosity for NDD-associated Trio variants - +/K1431M (ASD), +/K1918X (SCZ), and +/M2145T (bipolar disorder, BPD) - impact mouse behavior, brain development, and synapse structure and function. Heterozygosity for different Trio variants impacts motor, social, and cognitive behaviors in distinct ways that align with clinical phenotypes in humans. Trio variants differentially impact head and brain size with corresponding changes in dendritic arbors of motor cortex layer 5 pyramidal neurons (M1 L5 PNs). Although neuronal structure was only modestly altered in the Trio variant heterozygotes, we observe significant changes in synaptic function and plasticity. We also identified distinct changes in glutamate synaptic release in +/K1431M and +/M2145T cortico-cortical synapses. The TRIO K1431M GEF1 domain has impaired ability to promote GTP exchange on Rac1, but +/K1431M mice exhibit increased Rac1 activity, associated with increased levels of the Rac1 GEF Tiam1. Acute Rac1 inhibition with NSC23766 rescued glutamate release deficits in +/K1431M variant cortex. Our work reveals that discrete NDD-associated Trio variants yield overlapping but distinct phenotypes in mice, demonstrates an essential role for Trio in presynaptic glutamate release, and underscores the importance of studying the impact of variant heterozygosity in vivo.

Identification of SH2 Domain-Containing Protein 3C as a Novel, Putative Interactor of Dipeptidyl Peptidase 3.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent exopeptidase with broad specificity for four to eight amino acid residue substrates. It has a role in the regulation of oxidative stress response NRF2-KEAP1 pathway through the interaction with KEAP1. We have conducted stable isotope labeling by amino acids in a cell culture coupled to mass spectrometry (SILAC-MS) interactome analysis of TRex HEK293T cells using DPP3 as bait and identified SH2 Domain-Containing Protein 3C (SH2D3C) as prey. SH2D3C is one of three members of a family of proteins that contain both the SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain), named novel SH2-containing proteins (NSP). NSPs, including SH2D3C (NSP3), are adaptor proteins involved in the regulation of adhesion, migration, tissue organization, and immune response. We have shown that SH2D3C binds to DPP3 through its C-terminal Ras GEF-like domain, detected the colocalization of the proteins in living cells, and confirmed direct interaction in the cytosol and membrane ruffles. Computational analysis also confirmed the binding of the C-terminal domain of SH2D3C to DPP3, but the exact model could not be discerned. This is the first indication that DPP3 and SH2D3C are interacting partners, and further studies to elucidate the physiological significance of this interaction are on the way.

Modeling human mutations to understand TRIO GEF function during development

In a recent study, Bonnet and colleagues leveraged in silico structure prediction and human genetic data to understand the molecular regulation of the Rac1-activating guanie nucleotide exchange factor (Rac1-GEF) domain of Trio. Their work sheds new light on the role of Trio during axon guidance and explores the mechanism by which Trio GEF function is regulated in health and dysregulated in disease.

Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

Examination of clinically-derived p210 BCR/ABL1 RhoGEF mutations in a murine bone marrow transplantation model of CML

Expression of the p210 BCR/ABL1 fusion protein has been described in virtually all patients with chronic myelogenous leukemia (CML). Previous studies have identified a guanine nucleotide exchange factor (RhoGEF) domain within BCR that is retained in p210 BCR/ABL1. Missense mutations at residues T654 (T654K) and F547 (F547L) within this domain have been reported in a CML patient in blast crisis (BC). In this study, we have evaluated p210 BCR/ABL1 constructs that contain these substitutions in a murine bone marrow transplantation (BMT) model of CML. The mutants exhibit normal expression and tyrosine kinase activity but altered signaling. When examined in the BMT assay, mice that express the mutants exhibit earlier onset of disease but have significantly extended lifespans relative to mice that express unmodified p210 BCR/ABL1. While mice that express p210 BCR/ABL1 exhibit neutrophilia that progresses to a less differentiated phenotype at death, disease in the mutant mice is characterized by eosinophilia with no maturation arrest. This observation was confirmed in vitro using myeloid cells and was associated with enhanced p53 phosphorylation and G1/S arrest. These results suggest that residues within the RhoGEF domain of p210 BCR/ABL1 can influence disease progression.

Loss of the BCR-FGFR1 GEF Domain Suppresses RHOA Activation and Enhances B-Lymphomagenesis in Mice.

Transformation of hematopoietic stem cells by the BCR-FGFR1 fusion kinase found in a variant of stem cell leukemia/lymphoma (SCLL) syndrome leads to development of B-lymphomas in syngeneic mice and humans. In this study, we show that the relatively rapid onset of this leukemia is potentially related to oncogenic domains within the BCR component. BCR recruited a guanidine nucleotide exchange factor (GEF) domain to the fusion kinase to facilitate activation of small GTPases such as the Ras homology gene family, member A (RHOA). Deletion of this GEF domain increased leukemogenesis, enhanced cell survival and proliferation, and promoted stem cell expansion and lymph node metastasis. This suggests that, in an SCLL context, the presence of the endogenous GEF motif leads to reduced leukemogenesis. Indeed, loss of the GEF domain suppressed activation of RHOA and PTEN, leading to increased activation of AKT. Loss of the GEF domain enhanced cell proliferation and invasion potential, which was also observed in cells in which RHOA is knocked down, supported by the observation that overexpression of RHOA leads to reduced viability and invasion. In vivo depletion of RHOA in SCLL cells significantly increased disease progression and shortened latency. Collectively, these data show that the BCR GEF domain affects phenotypes associated with progression of SCLL through suppression of RHOA signaling. SIGNIFICANCE: RHOA activation is a critical event in the progression of BCR-FGFR1-driven leukemogenesis in stem cell leukemia and lymphoma syndrome and is regulated by the BCR GEF domain.

The role of Rho-GEF Trio in regulating tooth root development through the p38 MAPK pathway

Trio, the Rho guanine nucleotide exchange factor (Rho-GEF), plays diverse roles in cell migration, cell axon guidance and cytoskeleton reorganization. Conserved during evolution, Trio encodes two guanine nucleotide exchange factor domains (GEFs) and activates small GTPases. The Rho-family small GTPases RhoA and Rac1, which are target molecules of Trio, have been described to engage in craniofacial development and tooth formation. However, the exact role of Trio in tooth development remains elusive. In this study, we generated Wnt1-cre;Triofl/fl mice to address the potential function of Trio in tooth development. Wnt1-cre;Triofl/fl mice showed short root deformity as well as decreased expression of odontogenic makers such as RUNX2, OSX, OCN, and OPN. In vitro, Trio was silenced in human stem cells of dental papilla (SCAPs). Compared with the control group, the proliferation and migration ability in the experimental group was disrupted. After knocking down Trio in SCAPs, the cells showed phenotypes of poor odontogenic differentiation and weak mineralized nodules. To study the underlying mechanism, we investigated the p38 MAPK pathway and found that loss of Trio blocked the cascade transduction of p38 MAPK signaling. In conclusion, we identified Trio as a novel coordinator in regulating root development and clarified its relevant molecular events.

Neurodevelopmental disease-associated de novo mutations and rare sequence variants affect TRIO GDP/GTP exchange factor activity.

Bipolar disorder, schizophrenia, autism and intellectual disability are complex neurodevelopmental disorders, debilitating millions of people. Therapeutic progress is limited by poor understanding of underlying molecular pathways. Using a targeted search, we identified an enrichment of de novo mutations in the gene encoding the 330-kDa triple functional domain (TRIO) protein associated with neurodevelopmental disorders. By generating multiple TRIO antibodies, we show that the smaller TRIO9 isoform is the major brain protein product, and its levels decrease after birth. TRIO9 contains two guanine nucleotide exchange factor (GEF) domains with distinct specificities: GEF1 activates both Rac1 and RhoG; GEF2 activates RhoA. To understand the impact of disease-associated de novo mutations and other rare sequence variants on TRIO function, we utilized two FRET-based biosensors: a Rac1 biosensor to study mutations in TRIO (T)GEF1, and a RhoA biosensor to study mutations in TGEF2. We discovered that one autism-associated de novo mutation in TGEF1 (K1431M), at the TGEF1/Rac1 interface, markedly decreased its overall activity toward Rac1. A schizophrenia-associated rare sequence variant in TGEF1 (F1538Intron) was substantially less active, normalized to protein level and expressed poorly. Overall, mutations in TGEF1 decreased GEF1 activity toward Rac1. One bipolar disorder-associated rare variant (M2145T) in TGEF2 impaired inhibition by the TGEF2 pleckstrin-homology domain, resulting in dramatically increased TGEF2 activity. Overall, genetic damage to both TGEF domains altered TRIO catalytic activity, decreasing TGEF1 activity and increasing TGEF2 activity. Importantly, both GEF changes are expected to decrease neurite outgrowth, perhaps consistent with their association with neurodevelopmental disorders.

The Rho guanine nucleotide exchange factor (Rho-GEF) domain of a-kinase anchoring protein-13 (AKAP13) is necessary for optimal gonadotropin signaling in granulosa cells

AKAP13 plays an active role in gonadotropin-dependent gene transcription. We previously showed that AKAP13 augments cyclic AMP response element binding protein (CREB) activation in granulosa cells (GCs) and is required for optimal follicle stimulating hormone signaling. Since the exact mechanisms by which AKAP13 mediates gonadotropin signaling through CREB remains unclear, we sought to further explore regions of AKAP13 required for CREB activation.

RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.

RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.

Trio gene is required for mouse learning ability

Trio is a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Trio regulates cytoskeleton dynamics and actin remodeling and is involved in cell migration and axonal guidance in neuronal development. The null allele of the Trio gene led to embryonic lethality, and Trio null embryos displayed aberrant organization in several regions of the brain at E18.5, including hippocampus. Nestin-Trio−/− mice, in which the Trio gene was deleted specifically in the neuronal system by the Nestin-Cre system, displayed severe phenotypes, including low survival rate, ataxia and multiple developmental defects of the cerebellum. All Nestin-Trio−/− mice died before reaching adulthood, which hinders research on Trio gene function in adult mice. Thus, we generated EMX1-Trio−/− mice by crossing Trio-floxed mice with EMX1-Cre mice in which Cre is expressed in the brain cortex and hippocampus. EMX1-Trio−/− mice can survive to adulthood. Trio gene deletion results in smaller brains, an abnormal hippocampus and disordered granule cells in the dentate gyrus (DG) and cornu ammonis (CA). Behavior tests showed that Trio deletion interfered with the hippocampal-dependent spatial learning in the mice, suggesting that Trio plays critical roles in the learning ability of adult mice. We conclude that the Trio gene regulates the neuronal development of the hippocampus and that it affects the intelligence of adult mice.

Kalirin-9 and Kalirin-12 Play Essential Roles in Dendritic Outgrowth and Branching.

Proteins derived from the Kalrn gene, encoding 2 Rho guanine nucleotide exchange factor (GEF) domains, affect dendritic and axonal morphogenesis. The roles of endogenous Kalirin-9 (Kal9) and Kalirin-12 (Kal12), the Kalrn isoforms expressed before synaptogenesis, have not been studied in neurite growth and maturation during early development. The Caenorhabditis elegans and Drosophila melanogaster orthologues of Kalrn encode proteins equivalent to Kal9 but, lacking a kinase domain, neither organism expresses a protein equivalent to Kal12. Both in vivo and in vitro analyses of cortical neurons from total Kalrn knockout mice, lacking all major Kalirin isoforms, revealed a simplified dendritic arbor and reduced neurite length. Using isoform-specific shRNAs to reduce Kal9 or Kal12 expression in hippocampal cultures resulted in stunted dendritic outgrowth and branching in vitro, without affecting axonal polarity. Exposing hippocampal cultures to inhibitors of the first GEF domain of Kalirin (ITX3, Z62954982) blunted neurite outgrowth and branching, confirming its essential role, without altering the morphology of neurons not expressing Kalrn. In addition, exogenous expression of the active kinase domain unique to Kal12 increased neurite number and length, whereas that of the inactive kinase domain decreased neurite growth. Our results demonstrate that both endogenous Kal9 and endogenous Kal12 contribute to dendritic maturation in early development.

AMPylation Is Critical for Rab1 Localization to Vacuoles Containing Legionella pneumophila

ABSTRACTLegionella pneumophila is an intracellular pathogen that resides within a membrane-bound compartment that is derived from vesicles exiting the endoplasmic reticulum (ER). To create this compartment, these bacteria use a type IV secretion system to deliver effector proteins that subvert host cell functions. Several Legionella effector proteins modulate the function of the host protein Rab1, which is a GTPase that is recruited to the Legionella-containing vacuole (LCV). Here, we examined which of the Rab1-directed enzymatic activities displayed by Legionella effectors are important for localizing the Rab1 protein to the LCV membrane. The guanine nucleotide exchange factor (GEF) domain in the effector protein DrrA (SidM) was essential for Rab1 recruitment to the LCV and Rab1 AMPylation by the nucleotidyltransferase domain in DrrA was important for Rab1 retention. Legionella organisms producing mutant DrrA proteins that were severely attenuated for GEF activity in vitro retained the ability to localize Rab1 to the LCV. Rab1 localization to the LCV mediated by these GEF-defective mutants required AMPylation. Importantly, we found that efficient localization of Rab1 to the LCV occurred when Rab1 GEF activity and Rab1 AMPylation activity were provided by separate proteins. Rab1 phosphocholination (PCylation) by the effector protein AnkX, however, was unable to substitute for Rab1 AMPylation. Lastly, the defect in Rab1 localization to the LCV in AMPylation-deficient strains of Legionella was partially suppressed if the GTPase-activating protein (GAP) LepB was eliminated. Thus, our data indicate that AMPylation of Rab1 is an effective strategy to maintain this GTPase on the LCV membrane.

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.

SEC14 and Spectrin Domains 1 (Sestd1) and Dapper Antagonist of Catenin 1 (Dact1) Scaffold Proteins Cooperatively Regulate the Van Gogh-like 2 (Vangl2) Four-pass Transmembrane Protein and Planar Cell Polarity (PCP) Pathway during Embryonic Development in Mice

The planar cell polarity (PCP) pathway is a conserved non-canonical (β-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here, we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations, including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with Dact1, a knock-out mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.

AKAP13 Rho-GEF and PKD-binding domain deficient mice develop normally but have an abnormal response to β-adrenergic-induced cardiac hypertrophy.

BackgroundA-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.Methodology/Principal FindingsTo determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction.ConclusionsThese results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy.

Kalirin Promotes Neointimal Hyperplasia by Activating Rac in Smooth Muscle Cells

Kalirin is a multifunctional protein that contains 2 guanine nucleotide exchange factor domains for the GTPases Rac1 and RhoA. Variants of KALRN have been associated with atherosclerosis in humans, but Kalirin's activity has been characterized almost exclusively in the central nervous system. We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs). Kalirin-9 protein is expressed abundantly in aorta and bone marrow, as well as in cultured SMCs, endothelial cells, and macrophages. Moreover, arterial Kalirin was upregulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in 3 models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin Rho-guanine nucleotide exchange factor -1 inhibitor 1-(3-nitrophenyl)-1H-pyrrole-2,5-dione. With reduced Kalirin function, SMCs showed normal RhoA activation but diminished Rac1 activation, assessed as reduced Rac-GTP levels, p21-activated kinase autophosphorylation, and SMC migration. Kalrn(-/+) SMCs proliferated 30% less rapidly than wild-type SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ≈60% less in Kalrn(-/+) and SMC-specific Kalrn(-/+) mice than in control mice. Kalirin functions as a guanine nucleotide exchange factor for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.

Optical Control of Protein Activity by Fluorescent Protein Domains

Fluorescent proteins (FPs) are widely used as sensors of protein localization and activity, while other classes of light-absorbing proteins have been used for optogenetic control of proteins with light. Here we describe a previously unknown feature of a variant of the photochromic green FP Dronpa - the ability to undergo light-dependent dissociation and association, and use it to control protein activities with light. We created a fluorescent light-inducible protein (FLIP) design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains tetramerize and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. To our knowledge, this is the first case of a light-dependent interaction outside of natural light-responsive regulatory proteins, and this method also uniquely has self-reporting abilities. In ongoing work, we are examining the mechanism of caging by Dronpa domains in more detail and adapting the FLIP design to photochromic red FPs. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Optical Control of Protein Activity by Fluorescent Protein Domains. Xin X. Zhou. Biophys. J. 104(2)s: 680a. Full-Text PDF Open Archive

Echovirus 30 Induced Neuronal Cell Death through TRIO-RhoA Signaling Activation

BackgroundEchovirus 30 (Echo30) is one of the most frequently identified human enteroviruses (EVs) causing aseptic meningitis and encephalitis. However the mechanism underlying the pathogenesis of Echo30 infection with significant clinical outcomes is not completely understood. The aim of this investigation is to illustrate molecular pathologic alteration in neuronal cells induced by Echo30 infection using clinical isolate from young patient with neurologic involvement.Methodology/Principal FindingsTo characterize the neuronal cellular response to Echo30 infection, we performed a proteomic analysis based on two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrophotometric (MS) analysis. We identified significant alteration of several protein expression levels in Echo30-infected SK-N-SH cells. Among these proteins, we focused on an outstanding up-regulation of Triple functional domain (TRIO) in Echo30-infected SK-N-SH cells. Generally, TRIO acts as a key component in the regulation of axon guidance and cell migration. In this study, we determined that TRIO plays a role in the novel pathways in Echo30 induced neuronal cell death.Conclusions/SignificanceOur finding shows that TRIO plays a critical role in neuronal cell death by Echo30 infection. Echo30 infection activates TRIO-guanine nucleotide exchange factor (GEF) domains (GEFD2) and RhoA signaling in turn. These results suggest that Echo30 infection induced neuronal cell death by activation of the TRIO-RhoA signaling. We expect the regulation of TRIO-RhoA signaling may represent a new therapeutic approach in treating aseptic meningitis and encephalitis induced by Echo30.