Proteomics reveals reduced expression of transketolase in pyrimidine 5'-nucleotidase deficient patients.

PURPOSE: To date, it remains a challenge to correctly and timely diagnose red blood cell (RBC) enzymopathies that result in hereditary nonspherocytic hemolytic anemia (HNSHA), the third most common of which is pyrimidine 5'-nucleotidase (P5N) deficiency with just over 100 cases recognized and confirmed worldwide. EXPERIMENTAL DESIGN: We have investigated the RBC proteome of a patient with P5N deficiency due to a homozygous frameshift mutation in the NT5C3A gene. Protein expression levels were analyzed against healthy controls and against patients with hemolytic anemia of different origin, to account for the patient's elevated reticulocyte versus RBC ratio. RESULTS: Stringent relative quantification of the patient's protein levels revealed reduced levels of P5N, and unexpectedly, also decreased levels of transketolase, an enzyme involved in the nonoxidative phase of the pentose phosphate pathway, one of the few key pathways active in RBCs. Immunoblotting of whole blood samples from this and other P5N-deficient patients with dissimilar mutations indicated that P5N deficiency was correlated with reduced transketolase levels. CONCLUSIONS AND CLINICAL RELEVANCE: Consequently, insight into patient RBC proteomes illustrates potential benefit of coupling quantitative proteomics strategies with routine HNSHA diagnostic procedures. Proteomics facilitates finding novel biomarkers for HNSHA patients, for example, suffering from P5N deficiency, providing new prospects for future diagnosis and therapy.

Proteome adaptation of Saccharomyces cerevisiae to severe calorie restriction in Retentostat cultures.

Stationary-phase, carbon-starved shake-flask cultures of Saccharomyces cerevisiae are popular models for studying eukaryotic chronological aging. However, their nutrient-starved physiological status differs substantially from that of postmitotic metazoan cells. Retentostat cultures offer an attractive alternative model system in which yeast cells, maintained under continuous calorie restriction, hardly divide but retain high metabolic activity and viability for prolonged periods of time. Using TMT labeling and UHPLC-MS/MS, the present study explores the proteome of yeast cultures during transition from exponential growth to near-zero growth in severely calorie-restricted retentostats. This transition elicited protein level changes in 20% of the yeast proteome. Increased abundance of heat shock-related proteins correlated with increased transcript levels of the corresponding genes and was consistent with a strongly increased heat shock tolerance of retentostat-grown cells. A sizable fraction (43%) of the proteins with increased abundance under calorie restriction was involved in oxidative phosphorylation and in various mitochondrial functions that, under the anaerobic, nongrowing conditions used, have a very limited role. Although it may seem surprising that yeast cells confronted with severe calorie restriction invest in the synthesis of proteins that, under those conditions, do not contribute to fitness, these responses may confer metabolic flexibility and thereby a selective advantage in fluctuating natural habitats.

Challenges for red blood cell biomarker discovery through proteomics.

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties.

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

The diversity of protein turnover and abundance under nitrogen-limited steady-state conditions in Saccharomyces cerevisiae.

To establish more advanced models of molecular dynamics within cells, protein characteristics such as turnover rate and absolute instead of relative abundance have to be analyzed. We applied a proteomics strategy to analyze protein degradation and abundance in Saccharomyces cerevisiae. We used steady-state chemostat cultures to ascertain well-defined growth conditions and nitrogen limited media, which allowed us to rapidly switch from (14)N to (15)N-isotope containing media and to monitor the decay of the (14)N mono-isotope signals in time. We acquired both protein abundance information and degradation rates of 641 proteins. Half-lives of individual proteins were very diverse under nitrogen-limited steady-state conditions, ranging from less than 30 min to over 20 h. Proteins that act as single physical complexes do not always show alike half-lives. For example the chaperonin-containing TCP-1 complex showed similar intermediate half-lives ranging from 7 to 20 h. In contrast, the ribosome exhibited a wide diversity of half-lives ranging from 2.5 to over 20 h, although their cellular abundances were rather similar. The stabilities of proteins involved in the central sugar metabolism were found to be intermediary, except for the glycolytic enzymes Hxk1p and Fba1p and the TCA-cycle proteins Lsc2p and Kgd1p, which showed half-lives of over 20 h. These data stress the need for inclusion of quantitative data of protein turn-over rates in yeast systems biology.

Protein signatures associated with tumor cell dissemination in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. Strong prognostic indicators that predict development of distant metastases are the presence and number of lymph node metastases in the neck, and extranodal spread. Recently, it was shown in several studies that also the presence of disseminated tumor cells (DTC) in the bone marrow predicts development of distant metastases. We have investigated whether protein signatures could be detected in primary HNSCC that distinguish tumors that disseminate into the bone marrow from those that do not. Therefore, DTC-positive and -negative primary HNSCC tumors were analyzed by 2D-DIGE. A signature consisting of 51 differential protein spots was identified upon stratification for bone marrow status, which allowed a correct classification of DTC-positive and DTC-negative HNSCC tumors in 95% of cases, using hierarchical clustering. The most prominent feature within this signature was the down-regulation of CK19 in DTC-positive tumors. Our data show that tumor cell dissemination to the bone marrow, the onset of hematogenic metastasis, can be deduced from the protein profile in the primary tumor. The highly significant down-regulation of CK19 supports a model of epithelial-mesenchymal transition for tumors that show a high proclivity for hematogenic dissemination.

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels.

BACKGROUND: The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ. RESULTS: Comparing by proteomics WT and nat3Δ strains, using metabolic 15N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation. CONCLUSIONS: Protein expression levels change only marginally in between WT and nat3Δ. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in nat3Δ revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.

Protein biomarker discovery for head and neck cancer.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common cancer worldwide. Despite improvements in diagnosis and treatment, the five-year-survival rate of advanced HNSCC has only moderately increased, which is largely due to the high proportion of patients that present with advanced disease stage and the frequent development of relapse and second primary tumors. Protein biomarkers allowing early detection of primary HNSCC or relapse may aid to improve clinical outcome. Screening for precursor changes in the mucosal linings preceding the development of invasive tumors and for accurate prediction of risk of malignant transformation, may be propitious opportunities, which are as yet difficult. This review summarizes recent results in HNSCC proteomics for biomarker research. Despite the wide diversity of experimental designs, a few common markers have been detected. Although some of these potential biomarkers are very promising, they still have to be further clinically validated. Finally, treatment of advanced cancers of several sites within the head and neck has shifted significantly during the last decade, and also, targeted drugs have entered the clinic. This has major consequences for the research questions in HNSCC research and accordingly for the future direction of proteome research in HNSCC biomarker discovery.

Expression of the orphan protein Plet-1 during trichilemmal differentiation of anagen hair follicles.

The rat mAb 33A10 recognizes an antigen in a variety of mouse epithelial tissues. In this study, we investigated in detail the expression pattern of the 33A10-defined antigen in the hair follicle. We show that 33A10 reactivity is confined to the most differentiated keratinocytes of the outer root sheath (ORS), the companion layer (CL), and to cells of the sebaceous gland duct. In vitro, the 33A10-defined antigen is expressed in keratinocytes derived from the ORS and accumulates on induction of differentiation. Using microarray analysis and transient transfection approaches, we established that the 33A10-defined antigen is the orphan protein, Placenta-expressed transcript (Plet)-1. Biochemical data indicated that Plet-1 is a glycosylphosphatidylinositol-anchored glycoprotein with N-linked carbohydrates in addition to other posttranslational modifications. Although silencing of Plet-1 expression using stable RNA interference in ORS keratinocytes decreased cellular migration, it increased adhesion to collagens I and IV. Immunohistochemical analysis showed that Plet-1 was primarily localized at the leading edge of epidermal wounds, where keratinocytes contacted the eschar. The restricted localization in both differentiated ORS and CL cells contacting the hair fiber and epidermal wounds suggests a role for the Plet-1 protein in regulating the interaction of keratinocytes with inert tissues.

Exploring the membrane proteome--challenges and analytical strategies.

The analysis of proteins in biological membranes forms a major challenge in proteomics. Despite continuous improvements and the development of more sensitive analytical methods, the analysis of membrane proteins has always been hampered by their hydrophobic properties and relatively low abundance. In this review, we describe recent successful strategies that have led to in-depth analyses of the membrane proteome. To facilitate membrane proteome analysis, it is essential that biochemical enrichment procedures are combined with special analytical workflows that are all optimized to cope with hydrophobic polypeptides. These include techniques for protein solubilization, and also well-matched developments in protein separation and protein digestion procedures. Finally, we discuss approaches to target membrane-protein complexes and lipid-protein interactions, as such approaches offer unique insights into function and architecture of cellular membranes.

Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteome.

N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.

Quantitative erythrocyte membrane proteome analysis with Blue-native/SDS PAGE.

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient. This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.

Evaluation of cornulin, keratin 4, keratin 13 expression and grade of dysplasia for predicting malignant progression of oral leukoplakia.

Oral leukoplakia is defined as a white patch in the oral cavity that cannot be diagnosed as any other known disorder. These lesions carry an increased risk of malignant progression, and approximately 2-3% per year do progress to cancer. At present biopsies are histopathologically graded for dysplasia to assess the risk of progression, but this grading is somewhat subjective and of limited use for the individual patient. In a previous study we discovered by a comprehensive proteomics approach that compared to normal mucosa, protein expression of cornulin, keratin 4 and keratin 13 is decreased in tumors and severe dysplasia, preneoplastic tissue with a high risk of malignant progression. Here, we studied whether loss of expression of these proteins can predict malignant transformation of oral leukoplakia. Biopsies of 12 progressing and 36 non-progressing leukoplakia lesions were analyzed for cornulin, keratin 4 and keratin 13 expression by immunohistochemistry, and graded for dysplasia. Kaplan-Meier analysis showed that loss of expression of neither cornulin (p=0.075), keratin 4 (p=0.789) nor keratin 13 (p=0.732) was significantly associated with malignant transformation of leukoplakia lesions. However, decreased expression of these proteins was significantly associated with the presence of hyperkeratosis. Only dysplasia grading correlated significantly with malignant progression of leukoplakia (p=0.024). Despite the promising outlook that decreased cornulin, keratin 4 and keratin 13 expression in the oral mucosa is associated with a premalignant state, these markers do not predict malignant transformation of leukoplakia lesions. The most likely explanation is that the aberrant differentiation state of hyperkeratotic leukoplakia lesions already causes a decreased expression, obscuring the putative association with malignant transformation. Our results support the significance of dysplasia grading for the prediction of malignant transformation.

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; an unexpected effect of cardiolipin in alpha-ketoglutarate dehydrogenase and prohibitin complexes.

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Delta strain; taz1Delta strain; and acb1Deltataz1Delta strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains. Furthermore, a TAZ1ACB1 double deletion mutant strain was used in this study which has both a decrease in the length of the CL acyl chains and an increase in MLCL. BN/SDS PAGE analysis revealed that cardiolipin is important for the prohibitin-m-AAA protease complex, the alpha-ketoglutarate dehydrogenase complex and respiratory chain supercomplexes. The results indicate that the decreased level of complexes in taz1Delta and acb1Deltataz1Delta mitochondria is due to a decreased content of CL or the presence of MLCL.

Differential Proteomics Identifies Protein Biomarkers That Predict Local Relapse of Head and Neck Squamous Cell Carcinomas.

PURPOSE: The 5-year survival rates of head and neck squamous cell carcinomas (HNSCC) remain disappointing. HNSCCs develop in precursor fields of genetically altered cells that are often not completely resected when the tumor is excised, causing local relapse. These precursor fields are mostly recognized as dysplasia, but histologic grading cannot reliably predict malignant transformation. Our aim was to discover and validate protein biomarkers that can detect precursor fields and predict local relapse in HNSCC using immunostaining of surgical margins. EXPERIMENTAL DESIGN: We compared paired and genetically characterized normal, precursor, and tumor tissues of eight patients by proteome analysis to identify differentially expressed proteins. The prognostic value of candidate protein biomarkers was evaluated by immunohistochemical analysis of 222 surgical margins of 46 HNSCC patients who developed local relapse or remained disease free. Significant associations were determined by Kaplan-Meier survival analysis and Cox-proportional hazards models. RESULTS: Forty proteins showed significant differential expression (false discovery rate-corrected P < 0.05). Most discriminative markers suited for immunostaining were keratin 4 and cornulin. Low expression in the surgical margins of keratin 4 (hazard ratio, 3.8; P = 0.002), cornulin (hazard ratio, 2.7; P = 0.025), and their combination (hazard ratio, 8.8; P = 0.0005) showed a highly significant association with the development of local relapse. Dysplasia grading had no prognostic relevance. CONCLUSIONS: Immunohistochemical assessment of keratin 4 and cornulin expression in surgical margins of HNSCC patients outperforms histopathologic grading in predicting the risk for local relapse. These markers can be used to initiate more frequent and lifelong surveillance of patients at high risk of local relapse, and enable selection for adjuvant treatment or tertiary prevention trials. (Clin Cancer Res 2009;15(24):7666-75).

A three-way proteomics strategy allows differential analysis of yeast mitochondrial membrane protein complexes under anaerobic and aerobic conditions.

To investigate the effect of anaerobiosis on the Saccharomyces cerevisiae mitochondrial proteome and the formation of respiratory chain and other protein complexes, we analyzed mitochondrial protein extracts that were enriched from lysates of aerobic and anaerobic steady-state chemostat cultures. We chose an innovative approach in which native mitochondrial membrane protein complexes were separated by 1-D blue native PAGE, which was combined with quantitative analysis of each complex subunit using stable isotope labeling. LC-FT(ICR)-MS/MS analysis was applied to identify and quantify the mitochondrial proteins. In addition, to establish if changes in mitochondrial complex composition occurred under anaerobiosis, we investigated the 1-D blue native PAGE protein migration patterns by Pearson correlation analysis. Surprisingly, we discovered that under anaerobic conditions, where the yeast respiratory chain is not active, the respiratory chain supercomplexes, such as complex V dimer, complex (III)(2)(IV)(2) and complex (III)(2)(IV) were still present, although at reduced levels. Pearson correlation analysis showed that the composition of the mitochondrial complexes was unchanged under aerobic or anaerobic conditions, with the exception of complex II. In addition, this latter approach allowed screening for possible novel complex interaction partners, since for example protein Aim38p, with a yet unknown function, was identified as a possible component of respiratory chain complex IV.

Development of selective bisubstrate-based inhibitors against protein kinase C (PKC) isozymes by using dynamic peptide microarrays.

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP-binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate-based inhibitors that target the more selective peptide-binding site in addition to the ATP-binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate-based leads. Dynamic peptide microarrays were used to find peptide-binding site inhibitors. These were linked with chemoselective click chemistry to an ATP-binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate-based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCtheta amongst three isozymes.

Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer.

INTRODUCTION: We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients. METHODS: EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years. RESULTS: CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis). CONCLUSIONS: Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.

Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.