Role of Isoprenylcysteine Carboxylmethyltransferase-catalyzed Methylation in Rho Function and Migration

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoid-modified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase)² or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and methylated cysteine at its C terminus. Numerous studies have demonstrated that this post-translational processing not only facilitates protein association with cellular membranes, but also can play important roles in protein-protein interactions and protein stability (1, 6, 7). Thus, it is clear that CaaX processing is necessary for the biological activities of these proteins.The prenylation pathway has been targeted for potential anticancer therapy because most members of the Ras superfamily, which contains many known oncogenes, undergo CAAX processing. The Ras superfamily consists of five large subfamilies; the two most well-characterized are the Ras and Rho subfamilies (8). Both Ras and Rho proteins are processed by the CaaX pathway; Ras family members are farnesylated, while most Rho family members are geranylgeranylated. These monomeric GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. In their active states, Ras and Rho subfamily members control numerous cell signaling pathways that are involved in cell proliferation, differentiation, migration, polarity, and morphology (9).Abnormally high activity of Ras and Rho signaling pathways contribute to initiation and progression of many types of cancer (10, 11). For example, many breast cancers that are highly metastatic express abnormally high levels of Rho proteins (12). Rho proteins control migration and invasion of cells by tightly coordinating changes in the actin and microtubule cytoskeletons. Acting through their effectors, Rho proteins rearrange the actin cytoskeleton to respond to chemo-attractant gradients, polarize cells, and control migration and invasion. While cell migration is necessary for development, leukocyte function, and other normal cell biologies, dysregulation of migration and invasion results in cancer metastasis (13). Metastasis is an important and deadly progression of cancer and understanding the biology of migrating cancer cells is crucial for therapeutic targeting of this aspect of cancer.Pharmacologic targeting of the enzymes involved in the CaaX-processing pathway has emerged as a promising anticancer strategy. In particular, there has been much effort in designing inhibitors against the protein prenyltransferases, most notably FTase (14, 15). There is also recent evidence that inhibition of geranygeranylation of Rho proteins also impacts oncogenesis and metastasis (16–18). However, the overall success of the FTase inhibitors (FTIs) in the clinical setting has been somewhat disappointing. One possible reason is a phenomenon termed “alternate prenylation” in which some FTase substrates, most notably K- and N- Ras, are modified by GGTase and escape inhibition by FTIs (19–21). Because the Rce1 protease and Icmt methyltransferase act on all CaaX proteins, problems such as alternate prenylation would not arise if these enzymes were targeted. Hence, while protein prenyltransferase inhibitors still show some promise as anticancer agents, the emerging view that global attenuation of CaaX protein function may be advantageous in blocking cancer cell growth has increased interest in studying the two downstream enzymes involved in CaaX processing.While the biological consequences of prenylation are fairly well understood, the precise roles of C-terminal methylation in CaaX protein function are still elusive. Depending on the CaaX protein, methylation has been ascribed to roles in localization, protein-protein interactions and protein stability (11). The development of an Icmt knock-out mouse model has furthered our understanding of Icmt function (22, 23). Localization studies conducted in cells with genetically deleted Icmt have shown that methylation is important for proper membrane association of Ras proteins. However, the localization of Rho proteins in the absence of Icmt activity appears to be more complicated and may vary depending on family member and activation status (24–26). Importantly, inhibition of CaaX protein methylation via either genetic or pharmacologic targeting has shown a clear impact on oncogenic transformation and tumor growth (23, 27, 28).Defining the role of Icmt-mediated methylation in complex cellular behaviors such as migration and invasion is crucial for furthering our understanding of the impact of CaaX protein methylation on the biology of normal and cancer cells. In the current study, we have assessed the impact of Icmt inhibition on cell biological processes associated with the function of Rho proteins, specifically cell adhesion, morphology, and migration. We found that inhibition of Icmt results in a disruption of the actin cytoskeleton and impairs ligand-mediated activation of RhoA and Rac1, a potential consequence of increased RhoGDI binding to both RhoA and Rac1 when their methylation is impaired. Further, we show that the impact of Icmt inhibition on cell migration is due at least in part to impairment of RhoA and Rac1function. These findings establish a role for Icmt-mediated methylation in cell migration and further elucidate the role that methylation plays in the function of Rho GTPases.     

Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.     

Effect of solar particle event radiation on gastrointestinal tract bacterial translocation and immune activation

Space flight conditions within the protection of Earth's gravitational field have been shown to alter immune responses, which could lead to potentially detrimental pathology. An additional risk of extended space travel outside the Earth's gravitational field is the effect of solar particle event (SPE) radiation exposure on the immune system. Organisms that could lead to infection include endogenous, latent viruses, colonizing pathogenics, and commensals, as well as exogenous microbes present in the spacecraft or other astronauts. In this report, the effect of SPE-like radiation on containment of commensal bacteria and the innate immune response induced by its breakdown was investigated at the radiation energies, doses and dose rates expected during an extravehicular excursion outside the Earth's gravitational field. A transient increase in serum lipopolysaccharide was observed 1 day after irradiation and was accompanied by an increase in acute-phase reactants and circulating proinflammatory cytokines, indicating immune activation. Baseline levels were reestablished by 5 days postirradiation. These findings suggest that astronauts exposed to SPE radiation could have impaired containment of colonizing bacteria and associated immune activation.     

Species richness, environmental fluctuations, and temporal change in total community biomass

Theory and empirical results suggest that high biodiversity should often cause lower temporal variability in aggregate community properties such as total community biomass. We assembled microbial communities containing 2 to 8 species of competitors in aquatic microcosms and found that the temporal change in total community biomass was positively but insignificantly associated with diversity in a constant temperature environment. There was no evidence of any trend in variable temperature environments. Three non-exclusive mechanisms might explain the lack of a net stabilising effect of species richness on temporal change. (1) A direct destabilising effect of diversity on population level variances caused some populations to vary more when embedded in more diverse communities. (2) Similar responses of the different species to environmental variability might have limited any insurance effect of increased species richness. (3) Large differences in the population level variability of different species (i.e., unevenness) could weaken the relation between species richness and community level stability. These three mechanisms may outweigh the stabilising effects of increases in total community biomass with diversity, statistical averaging, and slightly more negative covariance in more diverse communities. Our experiment and analyses advocate for further experimental investigations of diversity-variability relations.     

The Proteasome Function Reporter GFPu Accumulates in Young Brains of the APPswe/PS1dE9 Alzheimer’s Disease Mouse Model

Alzheimer’s disease (AD), the most common cause of dementia, is neuropathologically characterized by accumulation of insoluble fibrous inclusions in the brain in the form of intracellular neurofibrillary tangles and extracellular senile plaques. Perturbation of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the pathogenesis of AD. However, studies on UPS functionality with various methods and AD models have achieved non-conclusive results. To get further insight into UPS functionality in AD, we have crossed a well-documented APPswe/PS1dE9 AD mouse model with a UPS functionality reporter, GFPu, mouse expressing green fluorescence protein (GFP) fused to a constitutive degradation signal (CL-1) that facilitates its rapid turnover in conditions of a normal UPS. Our western blot results indicate that GFPu reporter protein was accumulated in the cortex and hippocampus, but not striatum in the APPswe/PS1dE9 AD mouse model at 4 weeks of age, which is confirmed by fluorescence microscopy and elevated levels of p53, an endogenous UPS substrate. In accordance with this, the levels of ubiquitinated proteins were elevated in the AD mouse model. These results suggest that UPS is either impaired or functionally insufficient in specific brain regions in the APPswe/PS1dE9 AD mouse model at a very young age, long before senile plaque formation and the onset of memory loss. These observations may shed new light on the pathogenesis of AD.     

The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis

A modified latex agglutination (LA) test was compared with Gram-staining and counterimmunoelectrophoresis (CIE) for the rapid detection in the cerebrospinal fluid (CSF) of antigen to Haemophilus influenzae type b, Neisseria meningitidis groups A, B and C, Escherichia coli K1, Streptococcus pneumoniae and group B streptococci, seven frequent causes of bacterial meningitis in children. Of 50 CSF samples from patients with culture-proven bacterial meningitis 90% were correctly shown by the LA test to contain antigen of the responsible organism. Gram-staining revealed organisms in 80% of 45 of these samples. In 75% of the 40 samples that were of sufficient volume for CIE, positive results for the appropriate antigen were obtained. The concentration of antigen detected in the CSF by the LA test varied from undetectable to 800 000 ng/ml. Patients with a high concentration (more than 2000 ng/ml or a positive result at dilutions of CSF over 1/8) were significantly more likely to have a poor response to therapy (two died and two had persistent pleocytosis or bacteria in the CSF) than patients with a lower concentration (4/16 v. 0/18, P < 0.05). After appropriate therapy was begun the concentration of antigen fell dramatically, but measurable amounts of antigen persisted in the CSF for up to 6 days. The LA test detected bacterial antigen at concentrations 2 to 70 times below the lower limit detected by CIE. In seven additional patients who had received antibiotics before lumbar puncture was performed the LA test detected antigen from meningitis-causing bacteria even though cultures of the CSF were sterile. In another 145 patients who did not have meningitis the results of the LA test were negative. The LA test, done as described in this article, is easier to perform than CIE and should be a useful addition to the diagnostic tests carried out on the CSF of any patient suspected of having meningitis.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.