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Morphological and structural remodeling of the heart, including cardiac hypertrophy and fibrosis, has been considered as a therapeutic target for heart failure for approximately three decades. Groundbreaking heart failure medications demonstrating reverse remodeling effects have contributed significantly to medical advancements. However, nearly 50% of heart failure patients still exhibit drug resistance, posing a challenge to the healthcare system. Recently, characteristics of heart failure resistant to ARBs and β-blockers have been defined, highlighting preserved systolic function despite impaired diastolic function, leading to the classification of heart failure with preserved ejection fraction (HFpEF). The pathogenesis and aetiology of HFpEF may be related to metabolic abnormalities, as evidenced by its mimicry through endothelial dysfunction and excessive intake of high-fat diets. Our recent findings indicate a significant involvement of mitochondrial hyper-fission in the progression of heart failure. This mitochondrial pathological remodeling is associated with redox imbalance, especially hydrogen sulphide accumulation due to abnormal electron leak in myocardium. In this review, we also introduce a novel therapeutic strategy for heart failure from the current perspective of mitochondrial redox-metabolic remodeling. [ABSTRACT FROM AUTHOR]

The Ras family of small GTPases coordinates tissue development by modulating cell proliferation, cell-cell adhesion, and cellular morphology. Perturbations of any of these key steps alter nervous system development and are associated with neurological disorders. While the underlying causes are not known, genetic mutations in Ras and Rap GTPase signaling pathways have been identified in numerous neurodevelopmental disorders, including autism spectrum, neurofibromatosis, intellectual disability, epilepsy, and schizophrenia. Despite diverse clinical presentations, intersections between these two signaling pathways may provide a better understanding of how deviations in neurodevelopment give rise to neurological disorders. In this review, we focus on presynaptic and postsynaptic functions of Ras and Rap GTPases. We highlight various roles of these small GTPases during synapse formation and plasticity. Based on genomic analyses, we discuss how disease-related mutations in Ras and Rap signaling proteins may underlie human disorders. Finally, we discuss how recent observations have identified molecular interactions between these pathways and how these findingsmay provide insights into themechanisms that underlie neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

The Ras family of small GTPases coordinates tissue development by modulating cell proliferation, cell-cell adhesion, and cellular morphology. Perturbations of any of these key steps alter nervous system development and are associated with neurological disorders. While the underlying causes are not known, genetic mutations in Ras and Rap GTPase signaling pathways have been identified in numerous neurodevelopmental disorders, including autism spectrum, neurofibromatosis, intellectual disability, epilepsy, and schizophrenia. Despite diverse clinical presentations, intersections between these two signaling pathways may provide a better understanding of how deviations in neurodevelopment give rise to neurological disorders. In this review, we focus on presynaptic and postsynaptic functions of Ras and Rap GTPases. We highlight various roles of these small GTPases during synapse formation and plasticity. Based on genomic analyses, we discuss how disease-related mutations in Ras and Rap signaling proteins may underlie human disorders. Finally, we discuss how recent observations have identified molecular interactions between these pathways and how these findings may provide insights into the mechanisms that underlie neurodevelopmental disorders.

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify βPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of βPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, βPix is localized in the peripheral domain of the growth cone. Using βPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of βPix are specifically removed, we demonstrate that βPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in βPix NIKO mice, supporting the local action of βPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the βPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the βPix NIKO neurons. Altogether, these data indicate that βPix plays an important role in axonal regrowth during peripheral nerve regeneration. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Understanding of the evolution of metazoans from their unicellular ancestors is a fundamental question in biology. In contrast to fungi which utilize the Mon1-Ccz1 dimeric complex to activate the small GTPase RAB7A, metazoans rely on the Mon1-Ccz1-RMC1 trimeric complex. Here, we report a near-atomic resolution cryogenic-electron microscopy structure of the Drosophila Mon1-Ccz1-RMC1 complex. RMC1 acts as a scaffolding subunit and binds to both Mon1 and Ccz1 on the surface opposite to the RAB7A-binding site, with many of the RMC1-contacting residues from Mon1 and Ccz1 unique to metazoans, explaining the binding specificity. Significantly, the assembly of RMC1 with Mon1-Ccz1 is required for cellular RAB7A activation, autophagic functions and organismal development in zebrafish. Our studies offer a molecular explanation for the different degree of subunit conservation across species, and provide an excellent example of how metazoan-specific proteins take over existing functions in unicellular organisms. [ABSTRACT FROM AUTHOR]

When antigen-stimulated, mast cells release preformed inflammatory mediators stored in cytoplasmic granules. This occurs via a robust exocytosis mechanism termed degranulation. Our previous studies revealed that RhoA and Rac1 are activated during mast cell antigen stimulation and are required for mediator release. Here, we show that the RhoGEF, GEF-H1, acts as a signal transducer of antigen stimulation to activate RhoA and promote mast cell spreading via focal adhesion (FA) formation. Cell spreading, granule movement, and exocytosis were all reduced in antigen-stimulated mast cells when GEF-H1 was depleted by RNA interference. GEF-H1-depleted cells also showed a significant reduction in RhoA activation, resulting in reduced stress fiber formation without altering lamellipodia formation. Ectopic expression of a constitutively active RhoA mutant restored normal morphology in GEF-H1-depleted cells. FA formation during antigen stimulation required GEF-H1, suggesting it is a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 was activated by phosphorylation in conjunction with antigen stimulation. Syk kinase is linked to the FcεRI signaling pathway and the Syk inhibitor, GS-9973, blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis. We concluded that during FcεRI receptor stimulation, GEF-H1 transmits signals to RhoA activation and FA formation to facilitate the exocytosis mechanism. [ABSTRACT FROM AUTHOR]

Malignant cell plasticity is an important hallmark of tumor biology and crucial for metastasis and resistance. Cell plasticity lets cancer cells adapt to and escape the therapeutic strategies, which is the leading cause of cancer patient mortality. Epithelial cells acquire mobility via epithelial-mesenchymal transition (EMT), whereas mesenchymal cells enhance their migratory ability and clonogenic potential by acquiring amoeboid characteristics through mesenchymal-amoeboid transition (MAT). Tumor formation, progression, and metastasis depend on the tumor microenvironment (TME), a complex ecosystem within and around a tumor. Through increased migration and metastasis of cancer cells, the TME also contributes to malignancy. This review underscores the distinction between invasion pattern morphological manifestations and the diverse structures found within the TME. Furthermore, the mechanisms by which amoeboid-associated characteristics promote resistance and metastasis and how these mechanisms may represent therapeutic opportunities are discussed. In metastasis and resistance, cancer cells use the mesenchymal-amoeboid transition (MAT) to adapt. During this process, cancer cells switch from mesenchymal (elongated, matrix-degrading) to amoeboid (blebbed, rounded, contractility-driven, fast-moving) migration phenotypes. Cancer cells benefit from this adaptability by overcoming various microenvironmental challenges and facilitating invasion, dissemination, and survival (Created with BioRender.com). [Display omitted] • MAT, mesenchymal-amoeboid transition, enhances cancer metastasis and resistance. • Amoeboid phenotype: rounded shape, blebs, heightened actomyosin contractility, reduced adhesions. • Amoeboid cancer cells migrate faster than mesenchymal counterparts. • Amoeboid cancer cells exhibit stem-like properties and heightened treatment resistance. • Tumor microenvironment influences MAT, with decreased adhesion sites and increased confinement driving the transition. [ABSTRACT FROM AUTHOR]

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify βPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of βPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, βPix is localized in the peripheral domain of the growth cone. Using βPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of βPix are specifically removed, we demonstrate that βPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in βPix NIKO mice, supporting the local action of βPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the βPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the βPix NIKO neurons. Altogether, these data indicate that βPix plays an important role in axonal regrowth during peripheral nerve regeneration.

Ras GTPases are molecular switches that cycle between OFF and ON states depending on the bound nucleotide (i.e. GDP-bound and GTP-bound, respectively).The Rab GTPase, Sec4p, plays regulatory roles in multiple steps of intracellular vesicle trafficking. Nucleotide release is catalyzed by the Guanine Nucleotide Exchange Factor (GEF) Sec2p.Here, the integration of structural information with molecular dynamics (MD) simulations addressed a number of questions concerning the intrinsic and stimulated dynamics of Sec2p and Sec4p as well as the chain of structural deformations leading to GEF-assisted activation of the Rab GTPase.Sec2p holds an intrinsic ability to adopt the conformation found in the crystallographic complexes with Sec4p, thus suggesting that the latter selects and shifts the conformational equilibrium towards a pre-existing bound-like conformation of Sec2p.The anchoring of Sec4p to a suitable conformation of Sec2p favors the Sec2p-assisted pulling on itself of the α1/switch 1 (SWI) loop and of SWI, which loose any contact with GDP. Those deformations of Sec4p would occur earlier. Formation of the final Sec2p-Sec4p hydrophobic interface, accomplishes later. Disruption of the nucleotide cage would cause firstly loss of interactions with the guanine ring and secondly loss of interactions with the phosphates.The ease in sampling the energy landscape and adopting a bound-like conformation likely favors the catalyzing ability of GEFs for Ras GTPases.

The canonical process of activation of heterotrimeric G proteins by G protein coupled receptors (GPCRs) is well studied. Recently, a rapidly emerging paradigm has revealed the existence of a new, non-canonical set of cytosolic G protein modulators, guanine exchange modulators (GEMs). Among G proteins regulators, GEMs are uniquely capable of initiating pleiotropic signals: these bifunctional modulators can activate cAMP inhibitory (Gi) proteins and inhibit cAMP-stimulatory (Gs) proteins through a single short evolutionarily conserved module. A prototypical member of the GEM family, GIV/Girdin, integrates signals downstream of a myriad of cell surface receptors, e.g., growth factor RTKs, integrins, cytokine, GPCRs, etc., and translates these signals into G protein activation or inhibition. By their pleiotropic action, GIV and other GEMs modulate several key pathways within downstream signaling network. Unlike canonical G protein signaling that is finite and is triggered directly and exclusively by GPCRs, the temporal and spatial features of non-canonical activation of G protein via GIV-family of cytosolic GEMs are unusually relaxed. GIV uses this relaxed circuitry to integrate, reinforce and compartmentalize signals downstream of both growth factors and G proteins in a way that enables it to orchestrate cellular phenotypes in a sustained manner. Mounting evidence suggests the importance of GIV and other GEMs as disease modulators and their potential to serve as therapeutic targets; however, a lot remains unknown within the layers of the proverbial onion that must be systematically peeled. This perspective summarizes the key concepts of the GEM-dependent G protein signaling paradigm and discusses the multidisciplinary approaches that are likely to revolutionize our understanding of this paradigm from the atomic level to systems biology.

Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulated during mitophagy is not understood. Here, using a proximity-dependent biotinylation approach (miniTurbo), we identified C5orf51 as a specific interactor of GDP-locked RAB7A. C5orf51 also interacts with the RAB7A guanine nucleotide exchange factor (GEF) complex members MON1 and CCZ1. In the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised and the protein is degraded by the proteasome. Furthermore, depletion of C5orf51 also inhibited ATG9A recruitment to depolarized mitochondria. Together, these results indicate that C5orf51 is a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy. Abbreviations: ATG9A: autophagy related 9A; Baf A1: bafilomycin A1; BioID: proximity-dependent biotin identification; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CCZ1: CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; DQ-BSA: dye quenched-bovine serum albumin; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; KO: knockout; LRPPRC: leucine rich pentatricopeptide repeat containing; MG132: carbobenzoxy-Leu-Leu-leucinal; MON1: MON1 homolog, secretory trafficking associated; mtDNA: mitochondrial DNA; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RMC1: regulator of MON1-CCZ1; TBC1D15: TBC1 domain family member 15; TBC1D17: TBC1 domain family member 17; TOMM20: translocase of outer mitochondrial membrane 20; WDR91: WD repeat domain 91; WT: wild type. [ABSTRACT FROM AUTHOR]

Vav proteins belong to the class of guanine nucleotide exchange factors (GEFs) that catalyze the exchange of guanosine diphosphate (GDP) by guanosine triphosphate (GTP) on their target proteins. Here, especially the members of the small GTPase family, Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 homolog (Cdc42) can be brought into an activated state by the catalytic activity of Vav-GEFs. In the central nervous system (CNS) of rodents Vav3 shows the strongest expression pattern in comparison to Vav2 and Vav1, which is restricted to the hematopoietic system. Several studies revealed an important role of Vav3 for the elongation and branching of neurites. However, little is known about the function of Vav3 for other cell types of the CNS, like astrocytes. Therefore, the following study analyzed the effects of a Vav3 knockout on several astrocytic parameters as well as the influence of Vav3-deficient astrocytes on the dendritic development of cultured neurons. For this purpose, an indirect co-culture system of native hippocampal neurons and Vav3-deficient cortical astrocytes was used. Interestingly, neurons cultured in an indirect contact with Vav3-deficient astrocytes showed a significant increase in the dendritic complexity and length after 12 and 17 days in vitro (DIV). Furthermore, Vav3-deficient astrocytes showed an enhanced regeneration in the scratch wound heal assay as well as an altered profile of released cytokines with a complete lack of CXCL11, reduced levels of IL-6 and an increased release of CCL5. Based on these observations, we suppose that Vav3 plays an important role for the development of dendrites by regulating the expression and the release of neurotrophic factors and cytokines in astrocytes. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to evaluate changes in levels of adrenergic receptor β3 (ADRB3), Rho-related kinase 2 (ROCK2), and guanine nucleotide exchange factor (GEF), which play key roles in the adrenergic and cholinergic pathways of contraction-relaxation harmony in voiding physiology, and to explore the relationship between these proteins and overactive bladder (OAB). Methods: This study included 60 idiopathic OAB patients and a healthy control group. A validated OAB-validated 8 questionnaire was completed by all participants. Serum levels of ADRB3, ROCK2, and GEF were examined with enzyme-linked immunosorbent assays. Patient and control groups were compared in terms of these levels, and receiver operating characteristic (ROC) curves were generated for all parameters. Results: The levels of ROCK2 were significantly elevated, but there were no correlations between the OAB symptom score and the serum levels of ROCK2, ADRB3, and GEF in OAB patients. In the ROC analysis, ROCK2 alone provided the strongest potential relationship (area under the curve=0.651) with 84.9% sensitivity. The ROCK2+GEF combination provided a satisfactory relationship (AUC=0.755). The AUC for the ADRB3+ROCK2+GEF combination was 0.752, with 64.2% sensitivity and 88.2% specificity. Conclusions: The study results suggest that alterations in serum ROCK2 levels and the use of this parameter in combination with ADRB3 and GEF levels can shed light on the pathophysiology of idiopathic OAB syndrome and provide a new perspective for treatment. [ABSTRACT FROM AUTHOR]

Vav proteins belong to the class of guanine nucleotide exchange factors (GEFs) that catalyze the exchange of guanosine diphosphate (GDP) by guanosine triphosphate (GTP) on their target proteins. Here, especially the members of the small GTPase family, Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 homolog (Cdc42) can be brought into an activated state by the catalytic activity of Vav-GEFs. In the central nervous system (CNS) of rodents Vav3 shows the strongest expression pattern in comparison to Vav2 and Vav1, which is restricted to the hematopoietic system. Several studies revealed an important role of Vav3 for the elongation and branching of neurites. However, little is known about the function of Vav3 for other cell types of the CNS, like astrocytes. Therefore, the following study analyzed the effects of a Vav3 knockout on several astrocytic parameters as well as the influence of Vav3-deficient astrocytes on the dendritic development of cultured neurons. For this purpose, an indirect co-culture system of native hippocampal neurons and Vav3-deficient cortical astrocytes was used. Interestingly, neurons cultured in an indirect contact with Vav3-deficient astrocytes showed a significant increase in the dendritic complexity and length after 12 and 17 days in vitro (DIV). Furthermore, Vav3-deficient astrocytes showed an enhanced regeneration in the scratch wound heal assay as well as an altered profile of released cytokines with a complete lack of CXCL11, reduced levels of IL-6 and an increased release of CCL5. Based on these observations, we suppose that Vav3 plays an important role for the development of dendrites by regulating the expression and the release of neurotrophic factors and cytokines in astrocytes.

Purpose The aim of this study was to evaluate changes in levels of adrenergic receptor β3 (ADRB3), Rho-related kinase 2 (ROCK2), and guanine nucleotide exchange factor (GEF), which play key roles in the adrenergic and cholinergic pathways of contraction-relaxation harmony in voiding physiology, and to explore the relationship between these proteins and overactive bladder (OAB). Methods This study included 60 idiopathic OAB patients and a healthy control group. A validated OAB-validated 8 questionnaire was completed by all participants. Serum levels of ADRB3, ROCK2, and GEF were examined with enzyme-linked immunosorbent assays. Patient and control groups were compared in terms of these levels, and receiver operating characteristic (ROC) curves were generated for all parameters. Results The levels of ROCK2 were significantly elevated, but there were no correlations between the OAB symptom score and the serum levels of ROCK2, ADRB3, and GEF in OAB patients. In the ROC analysis, ROCK2 alone provided the strongest potential relationship (area under the curve=0.651) with 84.9% sensitivity. The ROCK2+GEF combination provided a satisfactory relationship (AUC=0.755). The AUC for the ADRB3+ROCK2+GEF combination was 0.752, with 64.2% sensitivity and 88.2% specificity. Conclusions The study results suggest that alterations in serum ROCK2 levels and the use of this parameter in combination with ADRB3 and GEF levels can shed light on the pathophysiology of idiopathic OAB syndrome and provide a new perspective for treatment.

When antigen-stimulated, mast cells release preformed inflammatory mediators stored in cytoplasmic granules. This occurs via a robust exocytosis mechanism termed degranulation. Our previous studies revealed that RhoA and Rac1 are activated during mast cell antigen stimulation and are required for mediator release. Here, we show that the RhoGEF, GEF-H1, acts as a signal transducer of antigen stimulation to activate RhoA and promote mast cell spreading via focal adhesion (FA) formation. Cell spreading, granule movement, and exocytosis were all reduced in antigen-stimulated mast cells when GEF-H1 was depleted by RNA interference. GEF-H1-depleted cells also showed a significant reduction in RhoA activation, resulting in reduced stress fiber formation without altering lamellipodia formation. Ectopic expression of a constitutively active RhoA mutant restored normal morphology in GEF-H1-depleted cells. FA formation during antigen stimulation required GEF-H1, suggesting it is a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 was activated by phosphorylation in conjunction with antigen stimulation. Syk kinase is linked to the FcεRI signaling pathway and the Syk inhibitor, GS-9973, blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis. We concluded that during FcεRI receptor stimulation, GEF-H1 transmits signals to RhoA activation and FA formation to facilitate the exocytosis mechanism.

Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gβγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis., (© 2024 The Author(s).)

The formation of nitrogen‐fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection. [ABSTRACT FROM AUTHOR]

Since the discovery by Madaule and Axel in 1985 of the first Ras homologue (Rho) protein in Aplysia and its human orthologue RhoB, membership in the Rho GTPase family has grown to 20 proteins, with representatives in all eukaryotic species. These GTPases are molecular switches that cycle between active (GTP bound) and inactivate (GDP bound) states. The exchange of GDP for GTP on Rho GTPases is facilitated by guanine exchange factors (GEFs). Approximately 80 Rho GEFs have been identified to date, and only a few GEFs associate with microtubules. The guanine nucleotide exchange factor H1, GEF-H1, is a unique GEF that associates with microtubules and is regulated by the polymerization state of microtubule networks. This review summarizes the regulation and functions of GEF-H1 and discusses the roles of GEF-H1 in human diseases. [ABSTRACT FROM AUTHOR]

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large‐scale phospho‐proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A‐S53D and eEF1A‐T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A‐S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange‐deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high‐throughput analysis are critical for eEF1A function. [ABSTRACT FROM AUTHOR]

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses.

Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development.

Activation of trimeric G proteins has been traditionally viewed as the exclusive job of G protein-coupled receptors (GPCRs). This view has been challenged by the discovery of non-receptor activators of trimeric G proteins. Among them, GIV (a.k.a. Girdin) is the first for which a guanine nucleotide exchange factor (GEF) activity has been unequivocally associated with a well defined motif. Here we discuss how GIV assembles alternative signaling pathways by sensing cues from various classes of surface receptors and relaying them via G protein activation. We also describe the dysregulation of this mechanism in disease and how its targeting holds promise for novel therapeutics.

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

Investigating cell migration in 3D settings has revealed that specific extracellular matrix environments require differential activities of the Rho GTPases for efficient migration. However, it is largely unknown how the activities of specific Rho GTPases are modulated to direct cell migration in response to different extracellular matrix cues. We have recently reported that extracellular matrix-dependent regulation of a specific Rho GEF is a fundamental mechanism governing cell migration in different microenvironments, providing a direct mechanism for extracellular matrix-specific regulation of Rho GTPase activity directing cell motility. We discovered that the Rho GEF βPix has a unique function during cell migration in fibrillar collagen environments by restraining RhoA signaling through a conserved signaling axis involving Cdc42 and the Rho GAP srGAP1. In this Commentary, we expand upon this new pathway and discuss potential mechanotransductive and therapeutic applications. Additionally, we speculate on a generalized role for Rho GEFs and GAPs in providing localized, context-dependent responses to the cellular microenvironment during cell migration and other cellular processes.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes. SYNOPSIS: The small GTPase Rab1 is a master regulator of both the early secretory and autophagic pathways, and is activated by the conserved multi‐subunit TRAPPIII complex. The cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to Rab1/Ypt1 provides a model for Rab1 activation on membranes. The cryo‐EM structure of the TRAPPIII‐Rab1/Ypt1 complex reveals the binding site for the Rab1/Ypt1 hypervariable domain.Stable membrane binding by the TRAPPIII complex is required for the activation of Rab1/Ypt1.A conserved amphipathic helix within the TRAPPIII regulatory subunit Trs85 is required for TRAPPIII membrane binding and activation of Rab1/Ypt1.Functional experiments suggest that Trs85‐mediated membrane anchoring of TRAPPIII is critical for both secretion and autophagy. [ABSTRACT FROM AUTHOR]

Although the small GTPase RAB37 acts as an organizer of autophagosome biogenesis, the upstream regulatory mechanism of autophagy via guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange in maintaining retinal function has not been determined. We found that retinitis pigmentosa GTPase regulator (RPGR) is a guanine nucleotide exchange factor that activates RAB37 by accelerating GDP-to-GTP exchange. RPGR directly interacts with RAB37 via the RPGR-RCC1-like domain to promote autophagy through stimulating exchange. Rpgr knockout (KO) in mice leads to photoreceptor degeneration owing to autophagy impairment in the retina. Notably, the retinopathy phenotypes of Rpgr KO retinas are rescued by the adeno-associated virus-mediated transfer of pre- trans -splicing molecules, which produce normal Rpgr mRNAs via trans- splicing in the Rpgr KO retinas. This rescue upregulates autophagy through the re-expression of RPGR in KO retinas to accelerate GDP-to-GTP exchange; thus, retinal homeostasis reverts to normal. Taken together, these findings provide an important missing link for coordinating RAB37 GDP-GTP exchange via the RPGR and retinal homeostasis by autophagy regulation. [Display omitted] • RPGR interacts with the small GTPase RAB37 via RLD • RPGR functions as a GEF for RAB37 GDP-GTP exchange • RPGR maintains autophagic homeostasis and retinal function • Photoreceptor degeneration owing to RPGR mutation can be cured by SMaRT The small GTPase RAB37 acts as an organizer for autophagosome biogenesis in a GTP-dependent manner. Ying et al. report that RPGR promotes autophagy by accelerating exchange from RAB37-GDP to -GTP, and further re-expression of RPGR by Rpgr PTM trans -splicing rescues photoreceptor degeneration via upregulation of autophagy. [ABSTRACT FROM AUTHOR]

Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the ability of this compound to modulate the activity of Epac and PKA, as determined by Rap1 activity and vasodilator-stimulated phosphoprotein phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that prevents the activation of Epac1 by cAMP.

The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2'-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (Rp)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.

Abnormally increased signaling by the GTPase RAP1 favors progression of diverse tumors. We have characterized the auto-regulation and activation of C3G (RAPGEF1), an activator of RAP1. This led us to discover mutations in non-Hodgkin’s lymphomas that activate C3G-RAP1 constitutively, suggesting that deregulation of C3G may favor the dissemination of tumor cells.

Rab35 is a small G protein involved in various cellular events including clathrin-dependent endocytosis, phagocytosis, and autophagy. DENND1B, a DENN family member, acts as a guanine nucleotide exchange factor (GEF) for Rab35 to convert it to the GTP-bound active form from the GDP-bound inactive form. DENND1B contains the DENN domain which harbors GEF activity for Rab35 in the N-terminus, while the clathrin binding motif and adaptor protein-2-interaction motif are at the C-terminus. In this study, we investigated the intracellular localization of DENN1B in various cell types and found novel DENND1B-localized gathered line structures in BS-C-1 cells and in some other cell types. The localization of DENND1B to gathered line structures was dependent on a specific region located in the C-terminus of DENND1B protein. DENND1B-localized gathered lines were partially associated with microtubules but not with F-actin; instead, F-actin bundles surrounded the assembly of gathered lines. We also show that the gathered line structures appeared at the bottom of spreading lamellipodia and disappeared at the retracting site during cell motility in EGF-stimulated BS-C-1 cells. These results shed light on a new role for DENND1B in the regulation of cell migration. [ABSTRACT FROM AUTHOR]

During development of the brain, neuronal circuits are formed through the projection of axons and dendrites in response to guidance signals. Rho GTPases (Rac1/RhoA/Cdc42) are major regulators of axo-dendritic outgrowth and guidance due to their role in controlling actin cytoskeletal dynamics, cell adhesion and motility. Functional redundancy of Rho GTPase-regulated pathways in neuronal development can mask the roles of specific GTPases. To examine potential Rho GTPase redundancy, we utilized a recently isolated hypomorphic mutation in a Caenorhabditis elegans Rac1 protein – CED-10(G30E) – which reduces the GTP binding and inhibits axon outgrowth of the PVQ interneurons. Here, we show that the CDC-42-specific guanine nucleotide exchange factor UIG-1 acts in parallel to CED-10/Rac1 to control PVQ axon outgrowth. UIG-1 performs this function in a cell-autonomous manner. Further, we found that transgenic expression of CDC-42 can compensate for aberrant CED-10(G30E)-regulated signalling during PVQ axon outgrowth. Together, our study reveals a previously unappreciated function for CDC-42 in PVQ axon outgrowth in C. elegans. [ABSTRACT FROM AUTHOR]

Insulin stimulates glucose uptake via the translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes. Several lines of evidence suggest that the small GTPase Rac1 plays an important role in insulin‐stimulated glucose uptake in skeletal muscle and adipocytes. The purpose of this study is to investigate the mechanisms whereby Rac1 is regulated in adipocyte insulin signaling. Here, we show that knockdown of the guanine nucleotide exchange factor FLJ00068 inhibits Rac1 activation and GLUT4 translocation by insulin and a constitutively activated form of the protein kinase Akt2. Furthermore, constitutively activated FLJ00068 induced Rac1 activation and Rac1‐dependent GLUT4 translocation. Collectively, these results suggest the involvement of FLJ00068 downstream of Akt2 in insulin‐stimulated glucose uptake signaling in adipocytes. [ABSTRACT FROM AUTHOR]

Rab GTPases play an important role in vesicle-mediated membrane trafficking in eukaryotes. Previous studies have demonstrated that deletion of RAB5/VPS21 reduces endocytosis and virulence of fungal phytopathogens in their host plants. However, Rab5 GTPase cycle regulators have not been characterized in Fusarium graminearum, the causal agent of Fusarium head blight (FHB) or head scab disease in cereal crops. In this study, we have identified and characterized a Rab5 guanine nucleotide exchange factor (GEF), the Vps9 homolog FgVps9, in F. graminearum. Yeast two hybrid (Y2H) assays have shown that FgVps9 specifically interacts with the guanosine diphosphate (GDP)-bound (inactive) forms of FgRab51 and FgRab52, the Rab5 isoforms in F. graminearum. Deletion of FgVPS9 shows impaired fungal growth and conidiation. Pathogenicity assays indicate that deletion of FgVPS9 can significantly decrease the virulence of F. graminearum in wheat. Cytological analyses have indicated that FgVps9 colocalizes with FgRab51 and FgRab52 on early endosomes and regulates endocytosis and autophagy processes. Gene expression and cytological examination have shown that FgVps9 and FgRab51 or FgRab52 function in concert to control deoxynivalenol (DON) biosynthesis by regulating the expression of trichothecene biosynthesis-related genes and toxisome biogenesis. Taken together, FgVps9 functions as a GEF for FgRab51 and FgRab52 to regulate endocytosis, which, as a basic cellular function, has significant impact on the vegetative growth, asexual development, autophagy, DON production, and plant infection in F. graminearum.

ABSTRACT Legionella pneumophila governs its interactions with host cells by secreting >300 different “effector” proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection. IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a “type IV” secretion system, which secretes more than 300 different “effector” proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

The Adenosine diphosphate-Ribosylation Factor (ARF) family belongs to the RAS superfamily of small GTPases and is involved in a wide variety of physiological processes, such as cell proliferation, motility and differentiation by regulating membrane traffic and associating with the cytoskeleton. Like other members of the RAS superfamily, ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. Several studies have reported that cancer cells are able to subvert membrane traffic regulators to enhance migration and invasion. Indeed, members of the ARF family, including ARF-Like (ARL) proteins have been implicated in tumorigenesis and progression of several types of cancer. Here, we review the role of ARF family members, their GEFs/GAPs and effectors in tumorigenesis and cancer progression, highlighting the ones that can have a pro-oncogenic behavior or function as tumor suppressors. Moreover, we propose possible mechanisms and approaches to target these proteins, toward the development of novel therapeutic strategies to impair tumor progression.

Rab GTPases play an important role in vesicle-mediated membrane trafficking in eukaryotes. Previous studies have demonstrated that deletion of RAB5 / VPS21 reduces endocytosis and virulence of fungal phytopathogens in their host plants. However, Rab5 GTPase cycle regulators have not been characterized in Fusarium graminearum , the causal agent of Fusarium head blight (FHB) or head scab disease in cereal crops. In this study, we have identified and characterized a Rab5 guanine nucleotide exchange factor (GEF), the Vps9 homolog FgVps9, in F. graminearum. Yeast two hybrid (Y2H) assays have shown that FgVps9 specifically interacts with the guanosine diphosphate (GDP)-bound (inactive) forms of FgRab51 and FgRab52, the Rab5 isoforms in F. graminearum. Deletion of FgVPS9 shows impaired fungal growth and conidiation. Pathogenicity assays indicate that deletion of FgVPS9 can significantly decrease the virulence of F. graminearum in wheat. Cytological analyses have indicated that FgVps9 colocalizes with FgRab51 and FgRab52 on early endosomes and regulates endocytosis and autophagy processes. Gene expression and cytological examination have shown that FgVps9 and FgRab51 or FgRab52 function in concert to control deoxynivalenol (DON) biosynthesis by regulating the expression of trichothecene biosynthesis-related genes and toxisome biogenesis. Taken together, FgVps9 functions as a GEF for FgRab51 and FgRab52 to regulate endocytosis, which, as a basic cellular function, has significant impact on the vegetative growth, asexual development, autophagy, DON production, and plant infection in F. graminearum. [ABSTRACT FROM AUTHOR]

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1‐month‐old Eif2b5I98M brain. Histological analyses indicated up‐regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751. [ABSTRACT FROM AUTHOR]

Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans -Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a , or RNAi of either Rab5a or GmVPS9s , significantly disrupted trafficking of mRFP–CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed. [ABSTRACT FROM AUTHOR]

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex. [ABSTRACT FROM AUTHOR]

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are one of the several families of cellular effectors of the prototypical second messenger cAMP. To understand the origin and molecular evolution of EPAC proteins, we performed a comprehensive phylogenetic analysis of EPAC1 and EPAC2. Our study demonstrates that unlike its cousin PKA, EPAC proteins are only present in multicellular Metazoa. Within the EPAC family, EPAC1 is only associated with chordates, while EPAC2 spans the entire animal kingdom. Despite a much more contemporary origin, EPAC1 proteins show much more sequence diversity among species, suggesting that EPAC1 has undergone more selection and evolved faster than EPAC2. Phylogenetic analyses of the individual cAMP binding domain (CBD) and guanine nucleotide exchange (GEF) domain of EPACs, two most conserved regions between the two isoforms, further reveal that EPAC1 and EPAC2 are closely clustered together within both the larger cyclic nucleotide receptor and RAPGEF families. These results support the notion that EPAC1 and EPAC2 share a common ancestor resulting from a fusion between the CBD of PKA and the GEF from RAPGEF1. On the other hand, the two terminal extremities and the RAS-association (RA) domains show the most sequence diversity between the two isoforms. Sequence diversities within these regions contribute significantly to the isoform-specific functions of EPACs. Importantly, unique isoform-specific sequence motifs within the RA domain have been identified.

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated 2-AG synthesis is known, our understanding of 2-AG release is limited. In NG108 cells and mouse midbrain tissue, we find that 2-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated 2-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated 2-AG release can occur via EVs.

Dendritic arbors are crucial for nervous system assembly, but the intracellular mechanisms that govern their assembly remain incompletely understood. Here, we show that the dendrites of PVD neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine-rich transmembrane (LRR-TM) receptor. DMA-1/LRR-TM interacts through a PDZ ligand motif with the guanine nucleotide exchange factor TIAM-1/GEF in a complex with act-4/Actin to pattern higher order 4° dendrite branches by localizing F-actin to the distal ends of developing dendrites. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. A partially redundant pathway, dependent on HPO-30/Claudin, regulates formation of 2° and 3° branches, possibly by regulating membrane localization and trafficking of DMA-1/LRR-TM. Collectively, our experiments suggest that HPO-30/Claudin localizes the DMA-1/LRR-TM receptor on PVD dendrites, which in turn can control dendrite patterning by directly modulating F-actin dynamics through TIAM-1/GEF.