Kai Stühler

Genetic analysis of the mouse brain proteome

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.

Identification and Functional Characterization of microRNAs Involved in the Malignant Progression of Gliomas

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma WHO grade III or secondary glioblastoma WHO grade IV. We explored the role of microRNAs (miRNAs) in glioma progression by investigating the expression profiles of 157 miRNAs in four patients with primary WHO grade II gliomas that spontaneously progressed to WHO grade IV secondary glioblastomas. Thereby, we identified 12 miRNAs (miR‐9, miR‐15a, miR‐16, miR‐17, miR‐19a, miR‐20a, miR‐21, miR‐25, miR‐28, miR‐130b, miR‐140 and miR‐210) showing increased expression, and two miRNAs (miR‐184 and miR‐328) showing reduced expression upon progression. Validation experiments on independent series of primary low‐grade and secondary high‐grade astrocytomas confirmed miR‐17 and miR‐184 as promising candidates, which were selected for functional analyses. These studies revealed miRNA‐specific influences on the viability, proliferation, apoptosis and invasive growth properties of A172 and T98G glioma cells in vitro. Using mRNA and protein expression profiling, we identified distinct sets of transcripts and proteins that were differentially expressed after inhibition of miR‐17 or overexpression of miR‐184 in glioma cells. Taken together, our results support an important role of altered miRNA expression in gliomas, and suggest miR‐17 and miR‐184 as interesting candidates contributing to glioma progression.

Difference gel electrophoresis.

Difference gel electrophoresis (DIGE) was invented to circumvent the inherent variability of 2‐DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15×20 cm slab of polyacrylamide gel. The originators of 2‐DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel‐to‐gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.

Detection of novel biomarkers of liver cirrhosis by proteomic analysis

Hepatic cirrhosis is a life‐threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4‐hydroxylase, tropomyosin, calponin, transgelin, and human microfibril–associated protein 4 (MFAP‐4). Tropomyosin, calponin, and transgelin reflect a contribution of activated stellate cells/myofibroblasts to chronic liver injury. The expression of tropomyosin, transgelin, and MFAP‐4, an extracellular matrix associated protein, were further evaluated by immunohistochemistry. Tropomyosin and MFAP‐4 demonstrated high serum levels in patients with hepatic cirrhosis of different causes. Conclusion: A quantitative analysis of MFAP‐4 serum levels in a large number of patients showed MFAP‐4 as novel candidate biomarker with high diagnostic accuracy for prediction of nondiseased liver versus cirrhosis [area under receiver operating characteristic curve (AUC) = 0.97, P < 0.0001] as well as stage 0 versus stage 4 fibrosis (AUC = 0.84, P < 0.0001), and stages 0 to 3 versus stage 4 fibrosis (AUC = 0.76, P < 0.0001). (HEPATOLOGY 2009.)

Study of Early Leaf Senescence in Arabidopsis thaliana by Quantitative Proteomics Using Reciprocal 14N/15N Labeling and Difference Gel Electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic 15N labeling followed by MS. Reciprocal 14N/15N labeling of entire Arabidopsis thaliana plants showed that full incorporation of 15N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and 15N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.

Proteome‐wide identification of mycobacterial pupylation targets

Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin‐like protein (PUP). In this study, we report a proteome‐wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine‐mediated pupylation acceptor sites was obtained by collision‐induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one‐third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.

Early leaf senescence is associated with an altered cellular redox balance in Arabidopsis cpr5/old1 mutants

Reactive oxygen species (ROS) are the inevitable by-products of essential cellular metabolic and physiological activities. Plants have developed sophisticated gene networks of ROS generation and scavenging systems. However, ROS regulation is still poorly understood. Here, we report that mutations in the Arabidopsis CPR5/OLD1 gene may cause early senescence through deregulation of the cellular redox balance. Genetic analysis showed that blocking stress-related hormonal signalling pathways, such as ethylene, salicylic acid, jasmonic acid, abscisic acid and sugar, did not affect premature cell death and leaf senescence. We took a bioinformatics approach and analysed publicly available transcriptome data of presymptomatic cpr5/old1 mutants. The results demonstrate that many genes in the ROS gene network show at least fivefold increases in transcripts in comparison with those of wild-type plants, suggesting that presymptomatic cpr5/old1 mutants are in a state of high-cellular oxidative stress. This was further confirmed by a comparative, relative quantitative proteomics study of Arabidopsis wild-type and cpr5/old1 mutant plants, which demonstrated that several Phi family members of glutathione s-transferases significantly increased in abundance. In summary, our genetic, transcriptomic and relative quantitative proteomics analyses indicate that CPR5 plays a central role in regulating redox balance in Arabidopsis.

Analysis of the Pancreatic Tumor Progression by a Quantitative Proteomic Approach and Immunhistochemical Validation.

To increase the knowledge about the development of pancreatic ductal adenocarcinoma, (PDAC) detailed analysis of the tumor progression is required. To identify proteins differentially expressed in the pancreatic intraepithelial neoplasia (PanIN), the precursor lesions of PDAC, we conducted a quantitative proteome study on microdissected PanIN cells. Proteins from 1000 microdissected cells were subjected to a procedure combining fluorescence dye saturation labeling with high resolution two-dimensional gel electrophoresis (2-DE). Differentially regulated protein spots were identified using protein lysates from PDAC tissues as a reference proteome followed by nanoLC-ESI-MS/MS. Thirty-seven single lesions of different PanIN grade (PanIN 1A/B, PanIN 2, PanIN 3) from nine patients were analyzed. Their protein expression was compared with each other, with PDAC cells and with normal ductal cells. The differential expression of differentially regulated protein spots was validated by means of immunohistochemistry using tissue microarrays. Of 2500 protein spots, 86 were found to be significantly regulated (p < 0.05, ratio > 1.6) during PanIN progression. Thirty-one nonredundant proteins were identified by mass spectrometry. Immunohistochemistry revealed that the differential expression of the selected candidate proteins major vault protein (MVP), anterior gradient 2 (AGR 2) and 14-3-3 sigma, annexin A4, and S100A10 could be successfully validated in PanIN lesions. The highly sensitive and robust proteome analysis revealed differentially regulated proteins involved in pancreatic tumor progression. The analysis of normal preneoplastic and neoplastic pancreatic tissue establishes a basis for identification of candidate biomarkers in PanIN progression that can be detected in pancreatic juice and in serum or are candidates for in vivo imaging approaches.

Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium

Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades.

Glutaredoxin regulates vascular development by reversible glutathionylation of sirtuin 1

Significance Embryonic development is one of the most amazing miracles in nature. The proteins and signaling events driving this highly complex process are far from being elucidated completely. For a long time, an important role of protein reduction and oxidation during development has been assumed. Here, we demonstrate the essential role of such a regulation during cardiovascular development: The modification of a single cysteine in the protein sirtuin 1 by the vertebrate-specific oxidoreductase glutaredoxin 2 is required for vessel formation and guidance. Our data indicate that this redox-signaling pathway based on glutaredoxin-dependent reversible S-glutathionylation may be also important for diseases of the cardiovascular system and pathological situations connected to angiogenesis, e.g., malignancies. Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione–disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD+-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.