Inequalities in cancer screening participation between adults with and without severe mental illness: results from a cross-sectional analysis of primary care data on English Screening Programmes.

BACKGROUND: People with severe mental illness (SMI) are 2.5 times more likely to die prematurely from cancer in England. Lower participation in screening may be a contributing factor. METHODS: Clinical Practice Research Datalink data for 1.71 million, 1.34 million and 2.50 million adults were assessed (using multivariate logistic regression) for possible associations between SMI and participation in bowel, breast and cervical screening, respectively. RESULTS: Screening participation was lower among adults with SMI, than without, for bowel (42.11% vs. 58.89%), breast (48.33% vs. 60.44%) and cervical screening (64.15% vs. 69.72%; all p < 0.001). Participation was lowest in those with schizophrenia (bowel, breast, cervical: 33.50%, 42.02%, 54.88%), then other psychoses (41.97%, 45.57%, 61.98%), then bipolar disorder (49.94%, 54.35%, 69.69%; all p-values < 0.001, except cervical screening in bipolar disorder; p-value > 0.05). Participation was lowest among people with SMI who live in the most deprived quintile of areas (bowel, breast, cervical: 36.17%, 40.23%, 61.47%), or are of a Black ethnicity (34.68%, 38.68%, 64.80%). Higher levels of deprivation and diversity, associated with SMI, did not explain the lower participation in screening. CONCLUSIONS: In England, participation in cancer screening is low among people with SMI. Support should be targeted to ethnically diverse and socioeconomically deprived areas, where SMI prevalence is greatest.

Haploinsufficiency of CYP8B1 associates with increased insulin sensitivity in humans.

BACKGROUNDCytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.METHODSTo determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.RESULTSMutation carriers had lower plasma 12α-hydroxylated/non-12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.CONCLUSIONOur findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.FUNDINGBiomedical Research Council/National Medical Research Council of Singapore.

Can proteomics elucidate mechanisms of antimicrobial resistance in Neisseria gonorrhoeae that whole genome sequencing is unable to identify? An analysis of protein expression within the 2016 WHO N. gonorrhoeae reference strains.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is of increasing concern. This study established a quantitative, scalable proteomics method to examine the WHO panel of N. gonorrhoeae isolates with completed closed genomic sequences and well-defined phenotypical and genotypical AMR patterns, to gain a greater understanding of AMR in N. gonorrhoeae. METHODS: 14 WHO reference strains were propagated, pooled stable isotope labelled lysates were used as an internal standard (IS). Protein lysates were mixed with IS, digested with trypsin and fractionated before analysis by nano-LC/MS/MS, in triplicate. The susceptible strain WHO F was used as reference to which the proteomic profiles of other strains were compared. Hierarchical clustering and permutation adjusted t-tests were performed to find proteins with significant fold changes. RESULTS: Standardised, reproducible protein expression profiles in N. gonorrhoeae reference strains were produced. Strains that have previously been shown to be highly similar using genomics, displayed different proteomic profiles. Several proteins from efflux pumps to stress responses, such as oxidative stress, toxin/antitoxin systems, were found to be altered in AMR strains. LtgE was upregulated in strains which displayed chromosomally mediated resistance to penicillin. MacB (the ATP hydrolysis part of macrolide efflux pump MacA-B), was ~twofold upregulated in WHO V (MIC of azithromycin >256 mg/L) and maybe associated with azithromycin resistance. CONCLUSIONS: A robust method was developed to study protein expression in N. gonorrhoeae. The proteome profiles could differentiate genetically similar stains. This study identified complex mechanisms in N. gonorrhoeae which may be associated with AMR.

Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson's disease.

Mutations in LRRK2, which encodes leucine-rich repeat kinase 2, are the most common genetic cause of familial and sporadic Parkinson's disease (PD), a degenerative disease of the central nervous system that causes impaired motor function and, in advanced stages, dementia. Dementia is a common symptom of another neurodegenerative disease, Alzheimer's disease, and research suggests that there may be pathophysiological and genetic links between the two diseases. Aggregates of ß amyloid [a protein produced through cleavage of amyloid precursor protein (APP)] are seen in both diseases and in PD patients carrying G2019S-mutant LRRK2. Using patient-derived cells, brain tissue, and PD model mice, we found that LRRK2 interacted with and phosphorylated APP at Thr668 within its intracellular domain (AICD). Phosphorylation of APP at Thr668 promoted AICD transcriptional activity and correlated with increased nuclear abundance of AICD and decreased abundance of a dopaminergic neuron marker in cultures and brain tissue. The AICD regulates the transcription of genes involved in cytoskeletal dynamics and apoptosis. Overexpression of AICD, but not a phosphodeficient mutant (AICDT668A), increased the loss of dopaminergic neurons in older mice expressing LRRK2G2019S Moreover, the amount of Thr668-phosphorylated APP was substantially greater in postmortem brain tissue and dopaminergic neurons (generated by reprogramming skin cells) from LRRK2G2019S patients than in those from healthy individuals. LRRK2 inhibitors reduced the phosphorylation of APP at Thr668 in the patient-derived dopaminergic neurons and in the midbrains of LRRK2G2019S mice. Thus, APP is a substrate of LRRK2, and its phosphorylation promotes AICD function and neurotoxicity in PD.

Discovery and characterisation of the automethylation properties of PRDM9.

We have previously characterised the histone lysine methyltransferase properties of PRDM9, a member of the PRDM family of putative transcriptional regulators. PRDM9 displays broad substrate recognition and methylates a range of histone substrates, including octamers, core histone proteins, and peptides. In the present study, we show that PRDM9 performs intramolecular automethylation on multiple lysine residues localised to a lysine-rich region on the post-SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain. PRDM9 automethylation is abolished by a single active-site mutation, C321P, also known to disrupt interactions with S-adenosylmethionine. We have taken an initial step towards tool compound generation through rational design of a substrate-mimic, peptidic inhibitor of PRDM9 automethylation. The discovery of automethylation in PRDM9 adds a new dimension to our understanding of PRDM9 enzymology.

Pseudomonas aeruginosa develops Ciprofloxacin resistance from low to high level with distinctive proteome changes.

Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. Different Ciprofloxacin concentrations selected different low level resistant mutants, and high level resistant mutants emerged from low level resistant mutants if stressed further by Ciprofloxacin. A deep quantitative proteomic study of the Ciprofloxacin resistant mutants uncovered the cellular pathways that supported such resistances. The two low level resistant mutants had different molecular mechanisms. One was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, and the other was probably due to iron and polyamine uptake and DNA repair. High level of resistance involved the mexCD-oprJ efflux pump and the downregulation of PQS quorum sensing. Other pathways might also have contributed to high level resistance, like the arginine deiminase pathway, catalase, peroxidase, protein degradation and DNA repair. The intracellular Ciprofloxacin concentration assay indicated that only the mexCD-oprJ overexpressed mutants had low drug accumulation. This study provided a comprehensive overview of the proteomic landscape in the evolution of Ciprofloxacin resistance in P. aeruginosa, and might have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa. Data are available via ProteomeXchange with identifier PXD004560. BIOLOGICAL SIGNIFICANCE: Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. We found that Ciprofloxacin resistance developed from low to high level. Two different low levels resistant molecular mechanisms were discovered from different mutants selected by different Ciprofloxacin concentrations, one was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, the other was probably due to iron, polyamine, and DNA repair. High level of Ciprofloxacin resistance all involved the efflux pump, mexCD-oprJ, and the downregulation of quorum sensing. The findings of this study provided insights into the evolution of Ciprofloxacin resistance in P. aeruginosa and should have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa.

Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.

CAPG and GIPC1: Breast Cancer Biomarkers for Bone Metastasis Development and Treatment.

BACKGROUND: Bone is the predominant site of metastasis from breast cancer, and recent trials have demonstrated that adjuvant bisphosphonate therapy can reduce bone metastasis development and improve survival. There is an unmet need for prognostic and predictive biomarkers so that therapy can be appropriately targeted. METHODS: Potential biomarkers for bone metastasis were identified using proteomic comparison of bone-metastatic, lung-metastatic, and nonmetastatic variants of human breast cancer MDA-MB-231 cells. Clinical validation was performed using immunohistochemical staining of tumor tissue microarrays from patients in a large randomized trial of adjuvant zoledronic acid (zoledronate) (AZURE-ISRCTN79831382). We used Cox proportional hazards regression, the Kaplan-Meier estimate of the survival function, and the log-rank test to investigate associations between protein expression, clinical variables, and time to distant recurrence events. All statistical tests were two-sided. RESULTS: Two novel biomarker candidates, macrophage-capping protein (CAPG) and PDZ domain-containing protein GIPC1 (GIPC1), were identified for clinical validation. Cox regression analysis of AZURE training and validation sets showed that control patients (no zoledronate) were more likely to develop first distant recurrence in bone (hazard ratio [HR] = 4.5, 95% confidence interval [CI] = 2.1 to 9.8, P < .001) and die (HR for overall survival = 1.8, 95% CI = 1.01 to 3.24, P = .045) if both proteins were highly expressed in the primary tumor. In patients with high expression of both proteins, zoledronate had a substantial effect, leading to 10-fold hazard ratio reduction (compared with control) for first distant recurrence in bone (P = .008). CONCLUSIONS: The composite biomarker, CAPG and GIPC1 in primary breast tumors, predicted disease outcomes and benefit from zoledronate and may facilitate patient selection for adjuvant bisphosphonate treatment.

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2-P4 study.

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 µM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.

Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 µM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

VHL-dependent regulation of a ß-dystroglycan glycoform and glycogene expression in renal cancer.

Identification of novel biomarkers and targets in renal cell carcinoma (RCC) remains a priority and one cellular compartment that is a rich potential source of such molecules is the plasma membrane. A shotgun proteomic analysis of cell surface proteins enriched by cell surface biotinylation and avidin affinity chromatography was explored using the UMRC2- renal cancer cell line, which lacks von Hippel-Lindau (VHL) tumour suppressor gene function, to determine whether proteins of interest could be detected. Of the 814 proteins identified ~22% were plasma membrane or membrane-associated, including several with known associations with cancer. This included ß-dystroglycan, the transmembrane subunit of the DAG1 gene product. VHL-dependent changes in the form of ß-dystroglycan were detected in UMRC2-/+VHL transfectants. Deglycosylation experiments showed that this was due to differential sialylation. Analysis of normal kidney cortex and conventional RCC tissues showed that a similar change also occurred in vivo. Investigation of the expression of genes involved in glycosylation in UMRC2-/+VHL cells using a focussed microarray highlighted a number of enzymes involved in sialylation; upregulation of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) was validated in UMRC2- cells compared with their +VHL counterparts and also found in conventional RCC tissue. These results implicate VHL in the regulation of glycosylation and raise interesting questions regarding the extent and importance of such changes in RCC.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion.

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.

A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top-down and bottom-up proteomic analyses.

Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2-D DIGE ('top-down') and LC-MS/MS ('bottom-up'). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high-throughput 1-D-LC-MS/MS approach, including known biomarkers and moderate-lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2-D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC-MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label-free analysis, greater run-to-run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1-D nano-LC-LTQ Orbitrap Velos analysis in a moderate through-put biomarker discovery process.

Shotgun proteomics of human bile in hilar cholangiocarcinoma.

The need to find biomarkers for hepatobiliary diseases including cholangiocarcinoma (CCA) has led to an interest in using bile as a proximal fluid in biomarker discovery experiments, although there are inherent challenges both in its acquisition and analysis. The study described here greatly extends previous studies that have started to characterise the bile proteome. Bile from four patients with hilar CCA was depleted of albumin and immunoglobulin G and analysed by GeLC-MS/MS. The number of proteins identified per bile sample was between 378 and 741. Overall, the products of 813 unique genes were identified, considerably extending current knowledge of the malignant bile proteome. Of these, 268 were present in at least 3 out of 4 patients. This data set represents the largest catalogue of bile proteins to date and together with other studies in the literature constitutes an important prelude to the potential promise of expression proteomics and subsequent validation studies in CCA biomarker discovery.

Peptide aptamer microarrays: bridging the bio-detector interface.

In the near future, personalised medicine and phase-0 trials will require that clinical practitioners move from the "one biomarker per disease" paradigm to the use of molecular signatures of disease for diagnosis and the prediction of a patient's response to treatment. These signatures will be composed of biomarkers specific to the disease, and will include over-expression of normal protein from a gene that does not carry a mutation; loss of expression of an essential protein; expression of a protein from a mutant gene; and metabolites whose levels are altered in disease. Surrogates for protein expression, such as alterations in the messenger RNA that encode for them, have already proved their value. The next challenge, then, in clinical biosensing is to enable the multiplexed detection of protein biomarkers, and perhaps the multiplexed detection of mixed biomarkers (metabolites, RNA and proteins) all in a single test. Given the plethora of available antibodies specific for biomarkers, why is this not already happening? We believe that the limitation lies in the nature of the antibody molecule itself, and especially the fact that antibodies have evolved to function in solution, while most diagnostic tests take place at a surface. We have accordingly turned to the design of alternative antibodies, and have identified a protein that appears to be unusually stable on surfaces. The new, non-antibody, scaffold protein is derived from human Stefin A, a natural inhibitor of the cathepsin family of proteases. We have engineered this protein so that it lacks natural binding partners, and introduced a series of new binding surfaces through randomisation or directed replacement of the surfaces used by Stefin A to bind to cathepsins. Our new probes show exquisite specificity and binding affinities comparable to antibodies, and can be used to probe biology in intact cells. More importantly, together and in collaboration with other groups in Chemistry or Engineering Departments, we have shown that these designer proteins can be used in optical detection of labelled target proteins from whole cell lysates in a highly multiplexed microarray format, as well as in label-free detection of unlabelled proteins using surface plasmon resonance, QCM, microcantilevers and using electrochemical assays on gold electrodes. We believe that the combination of optimised surface chemistry, robust and combinatorial designer biological probes and novel, robust and sensitive detection technologies will enable, in the near future, the introduction of multiplexed biomarker detection in the clinical setting, most likely in cancer where multiple biomarkers are known, but probes are still lacking.

Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations.

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10(-15)). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.

Differential effects of wild-type and A53T mutant isoform of alpha-synuclein on the mitochondrial proteome of differentiated SH-SY5Y cells.

Increased levels of wild-type (WT) alpha-synuclein (alpha-syn) and mutant A53T alpha-syn are associated with Parkinson's disease (PD), a disease linked to abnormal mitochondrial function. This study compared mitochondria prepared from differentiated SH-SY5Y cells overexpressing WT or A53T alpha-syn with control cells, using 2-D difference in-gel electrophoresis. Statistical analysis was carried out primarily using ANOVA (p < 0.01; Host:WT:A53T) and subsequently using independent t tests (host vs WT, host vs A53T). Of the protein spots found to be differentially expressed (n = 71; p < 0.01, >1.8/<-1.8 fold change), 63 proteins were identified by LC-MS/MS, with the majority (77%) significantly altered in WT samples only. Twenty-three proteins known to be integral components of the mitochondria were abnormally expressed including those with roles in ATP synthesis, oxidoreduction, motor activity, carbohydrate metabolism, protein transcription, and protein folding. Thirteen forms of cytoskeletal proteins were also found to be overexpressed in the mitochondrial preparations from WT alpha-syn cells, suggesting an increased interaction of mitochondria with the cytoskeletal network. Altered levels of four mitochondrial proteins (HSPA9 (mortalin), NDUFS1, DLAT, ATP5A1) were confirmed using Western blot analysis. Furthermore, a significant reduction in OXPHOS 1 activity was observed in the WT alpha-syn cells, suggesting that there are functional consequences of the observed altered protein expression changes in the mitochondria.

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.

Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel-Lindau transfectant cell line model.

The von Hippel-Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL-defective RCC cell line UMRC2 by transfection with vector control or VHL (-/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin-affinity chromatography and analysis by GeLC-MS/MS, VHL-associated changes in plasma membrane proteins were analysed. Comparative analysis of -/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the alpha 3 and beta1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL-defective cells. Western blotting confirmed these changes and also revealed VHL-dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient-matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL-dependent proteins that may be important in tumorigenesis.