Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.

CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The ∼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located ∼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.

The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27.

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.

Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway.

Vesicular pathways coupling the neuromuscular junction with the motor neuron soma are essential for neuronal function and survival. To characterize the organelles responsible for this long-distance crosstalk, we developed a purification strategy based on a fragment of tetanus neurotoxin (TeNT H(C)) conjugated to paramagnetic beads. This approach enabled us to identify, among other factors, the small GTPase Rab7 as a functional marker of a specific pool of axonal retrograde carriers, which transport neurotrophins and their receptors. Furthermore, Rab5 is essential for an early step in TeNT H(C) sorting but is absent from axonally transported vesicles. Our data demonstrate that TeNT H(C) uses a retrograde transport pathway shared with p75(NTR), TrkB, and BDNF, which is strictly dependent on the activities of both Rab5 and Rab7. Therefore, Rab7 plays an essential role in axonal retrograde transport by controlling a vesicular compartment implicated in neurotrophin traffic.

CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76.

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 muM at 37 degrees C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5.

The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily.

PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCepsilon at residue Ser(368) controls an established PKCepsilon scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCepsilon and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.

Proteomic evaluation of pathways associated with dexamethasone-mediated apoptosis and resistance in multiple myeloma.

We have used global protein expression analysis to characterize the pathways of dexamethasone-mediated apoptosis and resistance in myeloma. Analysis of MM.1S cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) identified a series of proteins that were up- and downregulated following dexamethasone treatment. Downregulated proteins included proteins involved in cell survival and proliferation, whereas upregulated proteins were involved in post-translational modification, protein folding and trafficking. A comparison with published gene expression studies identified FK binding protein 5 (FKBP5) (also known as FKBP51), a key regulatory component of the Hsp90-steroid-receptor complex to be increased at the mRNA and protein level postdexamethasone exposure. Quantitative real time polymerase chain reaction and 2D-PAGE analysis of the dexamethasone resistant cell line MM.1R demonstrated no increase in FKBP5, consistent with its association with dexamethasone-mediated apoptosis. Western blot analysis of FKBP5 and other members of the Hsp90-receptor complex showed an increase in FKBP5 whilst FKBP4 (also known as FKBP52) and Hsp90 expression remained constant. No changes were observed in MM.1R. In conclusion, we demonstrated that following steroid receptor signalling, the cell carries out a number of adaptive responses prior to cell death. Interfering with these adaptive responses may enhance the myeloma killing effect of dexamethasone.

Are parents and children satisfied with CAMHS?

BACKGROUND: Client Satisfaction with services is an important predictor of health care seeking, treatment compliance and health status outcome. Given that both parents and young people may have different views of services offered, this study examines both perspectives with respect to Child and Adolescent Mental Health Services (CAMHS) in Ireland. METHOD: Following ethical approval, the Client Satisfaction Questionnaire-8 (CSQ-8) were sent to all open cases attending three urban CAMHS. Returned questionnaires were received from 426 (280 parents and 146 adolescents) giving a response rate of 49%. RESULTS: No significant differences were observed between parents and adolescents regarding general satisfaction with CAMHS (Mann-Whitney U-test; Z=-0.255; p=0.799) with a range of good and excellent ratings between 77.1% (for the extent that program met user's needs) and 93.1% (for recommendation of program to a friend). Significant positive associations were found between age of child (Spearman's ρ=0.159; p=0.017), receiving a diagnosis (Mann-Whitney U-test; Z=-2.14; p=0.032), frequent attendance (χ 2=8.74; df=3; p=0.033) and living in close proximity to the service (χ 2=9.24; df=3; p=0.026). There was a strong negative correlation between reduction in impairment and levels of satisfaction (ρ=-0.44, n=275, p<0.000). Waiting time or duration in service were not associated with CSQ and 53% (145) requested clinic opening hours outside of 09:00 a.m. to 17:00 p.m. CONCLUSION: Regular user feedback, a robust and distinct measure of service quality, will ensure the development of effective, accessible, client-centered and responsive services, which can evolve in partnership with families and young people.

Androgen receptor is targeted to distinct subcellular compartments in response to different therapeutic antiandrogens.

PURPOSE: Antiandrogens are routinely used in the treatment of prostate cancer. Although they are known to prevent activation of the androgen receptor (AR), little is known about the mechanisms involved. This report represents the first study of the localization of wild-type AR following expression at physiologic relevant levels in prostate cells and treatment with androgen and antiandrogens. EXPERIMENTAL DESIGN: We have characterized a cellular model for prostate cancer using in situ cellular fractionation, proteomics, and confocal microscopy and investigated the effect of antiandrogens in clinical use on the subcellular localization of the AR. RESULTS: Different antiandrogens have diverse effects on the subcellular localization of the AR. Treatment with androgen results in translocation from the cytoplasm to the nucleoplasm, whereas the antiandrogens hydroxyflutamide and bicalutamide lead to reversible association with the nuclear matrix. In contrast, treatment with the antiandrogen cyproterone acetate results in AR association with cytoplasmic membranes and irreversible retention within the cytoplasm. In addition, we demonstrate that AR translocation requires ATP and the cytoskeleton, regardless of ligand. CONCLUSIONS: These results reveal that not all antiandrogens work via the same mechanism and suggest that an informed sequential treatment regime may benefit prostate cancer patients. The observed subnuclear and subcytoplasmic associations of the AR suggest new areas of study to investigate the role of the AR in the response and resistance of prostate cancer to antiandrogen therapy.

Regulation of the localisation and function of the oncogene LYRIC/AEG-1 by ubiquitination at K486 and K491.

The pivotal role of LYRIC/AEG-1 in malignant transformation, tumourigenesis and chemo-resistance has previously been demonstrated in different cell types and sub-cellular compartments. The localisation of LYRIC/AEG-1 appears crucial to its function and is regulated by three lysine-rich nuclear localisation signal regions, one of which was previously demonstrated to be modified by ubiquitin. Here we show that mutation of LYRIC/AEG-1 at K486 and K491 results in a loss of ubiquitination. A K486/491R double mutant that is incapable of ubiquitination shows reduced binding to the NFκB subunit p65 or importin-ß resulting in a distinctive peri-nuclear localisation of LYRIC/AEG-1. We also provide evidence to suggest that TOPORS, an E3 ligase that also regulates p53 modification may be responsible for LYRIC/AEG-1 ubiquitin modification. Overall we demonstrate that specific sites of LYRIC/AEG-1 ubiquitination are essential for regulating LYRIC/AEG-1 localisation and functionally interacting proteins.

A novel functional role for MMSET in RNA processing based on the link between the REIIBP isoform and its interaction with the SMN complex.

The chromosomal translocation t(4;14) deregulates MMSET (WHSC1/NSD2) expression and is a poor prognostic factor in multiple myeloma (MM). MMSET encodes two major protein isoforms. We have characterized the role of the shorter isoform (REIIBP) in myeloma cells and identified a clear and novel interaction of REIIBP with members of the SMN (survival of motor neuron) complex that directly affects the assembly of the spliceosomal ribonucleic particles. Using RNA-seq we show that REIIBP influences the RNA splicing pattern of the cell. This new discovery provides novel insights into the understanding of MM pathology, and potential new leads for therapeutic targeting.

Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations.

The maturation of human RET is adversely affected by a range of missense mutations found in patients with Hirschsprung's disease (HSCR), a complex multigenic disease. Here we show that two N-terminal cadherin-like domains, CLD1 and CLD2 (CLD(1-2)), from human RET adopt a clam-shell arrangement distinct from that of classical cadherins. CLD1 structural elements and disulfide composition are unique to mammals, indicating an unexpected structural diversity within higher and lower vertebrate RET CLD regions. We identify two unpaired cysteines that predispose human RET to maturation impediments in the endoplasmic reticulum and establish a quantitative cell-based RET maturation assay that offers a biochemical correlate of HSCR disease severity. Our findings provide a key conceptual framework and means of testing and predicting genotype-phenotype correlations in HSCR.

Proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8.

Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.

Labeling and identification of LNCaP cell surface proteins: a pilot study.

BACKGROUND: Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens. MATERIALS AND METHODS: Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin. Using the tag membrane proteins were purified and separated using two-dimensional gel electrophoresis and identified using mass spectrometry. E-cadherin and low density lipoprotein receptor (LDLR) were used as positive controls and also investigated following bicalutamide treatment. Membrane localization and androgen-regulation of proteins was confirmed using sub-cellular fractionation, Western blotting and microscopy. RESULTS: We have demonstrated efficient and specific protein biotinylation and purification of LNCaP plasma membrane proteins using Western analysis. E-cadherin and LDLR were regulated at the cell surface in response to R1881 and bicalutamide. Mass spectrometry identified several androgen-regulated membrane associated proteins including Prx-3 and GRP78 which are known to localize to other cellular compartments as well as the plasma membrane. We confirmed the localization of the identified proteins in LNCaP cells by co-localization with E-cadherin and immunohistochemistry of prostate tissue. CONCLUSION: Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples.

Structure and chemical inhibition of the RET tyrosine kinase domain.

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.

Proteomic analysis of primary cell lines identifies protein changes present in renal cell carcinoma.

New markers/targets for renal cell carcinoma (RCC) are needed to enable earlier detection and monitoring of disease and therapeutic targeting. To identify such molecules, normal and RCC-derived primary cell lines have been used as a simplified model system for studying changes that accompany tumorigenesis. Short-term cultures allow enrichment of relevant cell types from tissue samples, which is balanced against the potential for in vitro changes. Examination of 3 proteins with altered expression in RCC tissue showed the maintenance of normal-tumour differences in culture, although some changes were apparent, including alteration in the isoform of aldolase. Comparative analysis of primary cell lines by 2-DE found 43 proteins up-regulated and 29 down-regulated in at least three out of five tumour cell lines. Many of the observed changes have been previously reported in RCC, including up-regulation of several glycolytic enzymes, vimentin and heat shock protein 27, validating the approach. Additionally, several novel changes in protein expression were found, including up-regulation of several proteins involved in actin cytoskeleton organisation such as radixin and moesin, two members of the septin family, and the actin bundling protein, fascin. Validation studies using Western blotting and immunohistochemistry indicate that several of these molecules may be useful as markers for RCC.

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.

Identification of blottin: a novel gastric trefoil factor family-2 binding protein.

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes.

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases. This antiserum, termed PAP-1 (phospho antibody for proteomics-1), has proven useful for probing the serine phosphoproteome of antigen receptor-activated T lymphocytes. The present study shows that PAP-1 can also be used to explore serine kinases activated by cytokines and chemokines in T cells. Using PAP-1, together with proteomic analysis, the precursor form of the cytokine IL-16 (ProIL-16) was shown to be phosphorylated on Ser144 in antigen receptor-, SDF1alpha- and IL-2-activated T cells. Genetic and pharmacological-inhibitor experiments showed that the phosphorylation of ProIL-16 is dependent on activation of the kinases Erk1/2. IL-16 is secreted by mitogen-activated T cells, and the biochemical link between ProIL-16 and Erk1/2, revealed by studies with PAP-1, prompted analysis of the role of MAP kinases in this response. We show that TCR-mediated secretion of IL-16 is dependent on MAP kinases. The present study thus reveals how phosphoproteomic analysis opens previously unrecognized avenues for research, and yields novel insights about targets for MAP kinases in T lymphocytes.

Use of proteomic methodology for the characterization of human milk fat globular membrane proteins.

Characterization of the major human milk fat globular membrane proteins was carried out using proteomic techniques comprising two-dimensional polyacrylamide gel electrophoresis, followed by in situ PNGase F and trypsin digestion. Matrix-assisted laser desorption/ionization quadrupole time-of-flight and electrospray ionization mass spectrometry identified seven major protein components: alpha-lactalbumin, lysozyme precursor, beta-casein, clusterin, lactotransferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein. Sequence information on the protein-associated glycans was determined by matrix-assisted laser desorption-ionization quadrupole time-of-flight hybrid mass spectrometry. This glycan analysis revealed interesting fucosylation branching patterns which may be influential in maternal protection of the newborn against bacterial and viral pathogenic attack.