A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17

Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.

Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using Hollow Fibre Membrane Liquid-Phase Microextraction. This technique employs 2.5 cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC-MS/MS using positive mode electrospray ionisation with a 3.4 min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5 pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl
labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from
Gram-negative and Gram-positive bacteria. Our data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family

A predatory patchwork:membrane and surface structures of Bdellovibrio bacteriovorus

Predatory Bdellovibrio bacteriovorus bacteria are remarkable in that they attach to, penetrate and digest other Gram-negative bacteria, living and replicating within them until all resources are exhausted, when they escape the prey ghost to invade fresh prey. Remarkable remodeling of both predator and prey cell occurs during this process to allow the Bdellovibrio to exploit the intracellular niche they have worked so hard to enter, keeping the prey "bdelloplast" intact until the end of predatory growth. If one views motile non-predatory bacteria in a light microscope, one is immediately struck by how rare it is for bacteria to collide. This highlights how the cell surface of Bdellovibrio must be specialized and adapted to allow productive collisions and further to allow entry into the prey periplasm and subsequent secretion of hydrolytic enzymes to digest it. Bdellovibrio can, however, also be made to grow artificially without prey; thus, they have a large genome containing both predatory genes and genes for saprophytic heterotrophic growth. Thus, the membrane and outer surface layers are a patchwork of proteins encompassing not only those that have a sole purpose in heterotrophic growth but also many more that are specialized or employed to attach to, enter, remodel, kill and ultimately digest prey cells. There is much that is as yet not understood, but molecular genetic and post-genomic approaches to microbial physiology have enhanced the pioneering biochemical work of four decades ago in characterizing some of the key events and surface protein requirements for prey attack.

The role of secreted frizzled-related protein 2 expression in prostate cancer

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2.

METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients.

CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

BAR domains as sensors of membrane curvature:the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.