Rafael Rojas

Gαq Directly Activates p63RhoGEF and Trio via a Conserved Extension of the Dbl Homology-associated Pleckstrin Homology Domain

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Gα12/13 and Gαq/11 families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Gα12/13-mediated RhoA activation, proteins that functionally couple Gαq/11 to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Gαq/11 signaling to RhoA based on its ability to synergize with Gαq/11 resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Gαq directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Gαq relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Gαq.

Multifunctional Roles for the PH Domain of Dbs in Regulating Rho GTPase Activation

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivodespite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.

Ras-mediated loss of the pro-apoptotic response protein Par-4 is mediated by DNA hypermethylation through Raf-independent and Raf-dependent signaling cascades in epithelial cells

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Effects of dietary glycemic index on brain regions related to reward and craving in men

BACKGROUND Qualitative aspects of diet influence eating behavior, but the physiologic mechanisms for these calorie-independent effects remain speculative. OBJECTIVE We examined effects of the glycemic index (GI) on brain activity in the late postprandial period after a typical intermeal interval. DESIGN With the use of a randomized, blinded, crossover design, 12 overweight or obese men aged 18-35 y consumed high- and low-GI meals controlled for calories, macronutrients, and palatability on 2 occasions. The primary outcome was cerebral blood flow as a measure of resting brain activity, which was assessed by using arterial spin-labeling functional magnetic resonance imaging 4 h after test meals. We hypothesized that brain activity would be greater after the high-GI meal in prespecified regions involved in eating behavior, reward, and craving. RESULTS Incremental venous plasma glucose (2-h area under the curve) was 2.4-fold greater after the high- than the low-GI meal (P = 0.0001). Plasma glucose was lower (mean ± SE: 4.7 ± 0.14 compared with 5.3 ± 0.16 mmol/L; P = 0.005) and reported hunger was greater (P = 0.04) 4 h after the high- than the low-GI meal. At this time, the high-GI meal elicited greater brain activity centered in the right nucleus accumbens (a prespecified area; P = 0.0006 with adjustment for multiple comparisons) that spread to other areas of the right striatum and to the olfactory area. CONCLUSIONS Compared with an isocaloric low-GI meal, a high-GI meal decreased plasma glucose, increased hunger, and selectively stimulated brain regions associated with reward and craving in the late postprandial period, which is a time with special significance to eating behavior at the next meal. This trial was registered at clinicaltrials.gov as NCT01064778.

Neuroimaging in drug and substance abuse part II: Opioids and solvents

The central nervous system is one of the primary targets for the detrimental effects of drugs of abuse. Diagnostic imaging, especially MRI, plays an important role in the detection of complications associated with drug abuse. We present the imaging findings associated with the abuse of opioids and other morphine derivatives, as well, as solvents. Of the morphine derivatives, heroin is the most commonly abused. Several CNS pathologic effects have been described in association with its abuse. These include neurovascular complications such as microvascular ischemic changes or ischemic stroke. A rare form of leukoencephalopathy has been described in those abusers who inhale heroin vapors. Other neurologic complications include atrophy and various infectious processes. Solvent inhalation is a common practice among adolescents and young adults secondary to its ease of access and low cost. The most important component of industrial solvents is toluene. Complications of toluene abuse may be either acute, showing no neuroradiological changes, or chronic, characterized by cerebral and cerebellar demyelination as well as atrophy.

Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases

Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases.

SmgGDS Is a Guanine Nucleotide Exchange Factor That Specifically Activates RhoA and RhoC

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.

Magnetic resonance imaging findings in substance abuse: Alcohol and alcoholism and syndromes associated with alcohol abuse

Alcohol abuse is common among the population and results in significant diseases that shorten life span. Ethanol may result in chronic brain changes such as atrophy but may also result in neurologic disease that may be acute or chronic and sometimes life threatening. Accompanying vitamin deficiencies may lead to Wernickes encephalopathy and changes in serum osmosis may lead to several acute demyelinating disorders. In addition, pregnant women who consume alcohol place their babies at high risk for the fetal alcohol syndrome. In this article we review these disorders and emphasize their imaging features.

Brain responses to food images during the early and late follicular phase of the menstrual cycle in healthy young women: relation to fasting and feeding

BACKGROUND Food intake fluctuates throughout the menstrual cycle; it is greater during the early follicular and luteal phases than in the late follicular (periovulatory) phase. Ovarian steroids can influence brain areas that process food-related information, but the specific contribution of individual hormones and the importance of the prandial state remain unknown. OBJECTIVE The objective was to examine whether brain activation during food visualization is affected by changes in estradiol concentration in the fasted and fed conditions. DESIGN Nine eumenorrheic, lean young women [mean (±SD) age: 26.2 ± 3.2 y; body mass index (in kg/m(2)): 22.4 ± 1.2] completed 2 visits, one in the early (low estradiol) and one in the late (high estradiol) follicular phase of their menstrual cycle. At each visit, subjects underwent functional magnetic resonance imaging while they viewed food and nonfood images, before and after a standardized meal. Region-of-interest analysis was used to examine the effect of follicular phase and prandial state on brain activation (food > nonfood contrast) and its association with estradiol concentration. RESULTS Differences were identified in the inferior frontal and fusiform gyri. In these areas, visualization of food elicited greater activation in the fed state than during fasting but only in the late follicular phase, when estradiol concentration was high. The change in estradiol concentration across the follicular phase (late minus early) was inversely correlated with the change in fusiform gyrus activation in the fasted state but not in the fed state. CONCLUSION Our findings suggest that estradiol may reduce food intake by decreasing sensitivity to food cues in the ventral visual pathway under conditions of energy deprivation. This trial was registered at clinicaltrials.gov as NCT00130117.

High-Throughput Screening for Small Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY—Texas Red—GTPγS (BODIPY-TR-GTPγS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [ 35S] GTPγS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [ 35S] GTPγS binding but had little to no effect on RhoA or Gα o [35S] GTPγS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications. (Journal of Biomolecular Screening 2009:161-172)