Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins.

Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.

Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

A practical guide to the ICPL_ESIQuant software for ICPL-based quantitative proteomics.

ICPL_ESIQuant is a proteomics software tool for quantitatively analyzing large mass spectrometric datasets acquired from ICPL based proteomics experiments. It is able to process mass spectrometric data from various vendors and implements results from the Mascot search engine to generate protein and peptide result tables. This protocol briefly introduces ICPL_ESIQuant and presents a detailed step by step tutorial, how to use the software with MS datasets obtained from ICPL duplex, triplex and quadruplex experiments. Requiring MS raw data in .mzXML file format and Mascot search results in .dat format as input, ICPL_ESIQuant reliably quantifies ICPL labeled proteins and provides additional information about all detected, sequenced and identified features in the sample. The software supports both the shotgun and the directed proteomics strategy, enabling the user to apply mass inclusion lists for identifying peptides not fragmented in the first MS cycle. The software together with a test dataset is freely available under http://sourceforge.net/projects/icplquant/. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.

Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 "ex vivo". The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 µm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 µm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.

Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides.

The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a-mediated formation of singlet oxygen ((1)O(2)). Exposure to (1)O(2) induces the alternative sigma factors RpoE and RpoH(II) which then promote transcription of photooxidative stress-related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base-pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to (1)O(2). The higher (1)O(2) sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoH(II)-dependent genes. We used co-immunoprecipitation of FLAG-tagged Hfq combined with RNA-seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, > 70% of the Hfq-bound sRNAs are (1)O(2)-affected. Proteomics analysis of the Hfq-deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.

Anoxygenic photosynthesis and photooxidative stress: a particular challenge for Roseobacter.

Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O2) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O2 compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O2 during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O2 stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O2 generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O2 in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O2, most likely a consequence of their different lifestyles.

Evaluation of protein loading techniques and improved separation in OFFGEL isoelectric focusing.

2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.

Systematic comparative protein expression profiling of clear cell renal cell carcinoma: a pilot study based on the separation of tissue specimens by two-dimensional gel electrophoresis.

Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine.

Equalizer technology--Equal rights for disparate beads.

One major limitation in proteomics is the detection and analysis of low-abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low-abundant proteins was introduced by Boschetti and co-workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 butyl, FP-HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role.

ICPLQuant - A software for non-isobaric isotopic labeling proteomics.

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.

Acidianus, Sulfolobus and Metallosphaera surface layers: structure, composition and gene expression.

The cell walls of Sulfolobales species consist of proteinaceous S-layers assembled from two polypeptides, SlaA and SlaB. We isolated the large S-layer protein of Acidianus ambivalens and both S-layer subunits of Sulfolobus solfataricus and Metallosphaera sedula, respectively. The slaAB genes, lying adjacently in the chromosomes, are constitutively transcribed as bicistronic operons in A. ambivalens and S. solfataricus. A smaller slaA transcript appeared in Northern hybridizations of A. ambivalens RNA. PCRs experiments showed that 80-85% of the transcripts stop at an oligo-T terminator downstream of slaA while 15-20% are read through to a second terminator downstream of slaB. The bicistronic operons including promoter and terminator regions are conserved in the Sulfolobales. While no SlaA homologue is found outside the Sulfolobales, SlaB is distantly similar to S-layer proteins of other Crenarchaeota, e.g. the Staphylothermus marinus tetrabrachion. Molecular modelling suggests SlaBs to be composed of 2-3 consecutive beta sandwich domains, a coiled-coil domain of 15-17 nm in length and a C-terminal transmembrane helix. Electron microscopy shows crystalline protein arrays with triangular and hexagonal pores. We propose that the mushroom-shaped 'unit cells' of the Sulfolobales' S-layers consist of three SlaBs anchoring the complex in the membrane and six SlaAs forming the detergent-resistant outer sacculus.

Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

Hemorphins act as homeostatic agents in response to endotoxin-induced stress.

The effect of synthetic LVV-hemorphin-7 and hemorphin-7 on hypothalamo-pituitary-adrenocortical axis activity in response to endotoxin-induced stress was studied. The intraperitoneal (ip) endotoxin (lipopolysaccaride, LPS) (0.5 mg/kg) administration in combination with hemorphin (1 mg/kg) induce significant decrease in plasma corticosterone and modest decrease in plasma levels of tumor necrosis factor-alpha (TNFalpha) in compare with elevated levels of both corticosterone and TNFalpha in plasma of rats received LPS administration alone. Increased activity of calcineurin in both plasma and brain of rats received ip administration of LPS, was recovered under LPS + hemorphin treatment. In two independent proteome analysis, using 2-dimensional fluorescence difference gel electrophoresis and the isotope coded protein label technology, peptidyl-prolyl cis-trans-isomerase A (cyclophilin A) was identified as regulated by hemorphins protein in mouse brain. A therapeutic potential of hemorphins and mechanisms of their homeostatic action in response to endotoxin-induced stress are discussed.

Involvement of a Golgi-resident GPI-anchored protein in maintenance of the Golgi structure.

The Golgi apparatus consists of a series of flattened cisternal membranes that are aligned in parallel to form stacks. Cytosolic-oriented Golgi-associated proteins have been identified that may coordinate or maintain the Golgi architecture. Here, we describe a novel GPI-anchored protein, Golgi-resident GPI-anchored protein (GREG) that has a brefeldin A-sensitive Golgi localization. GREG resides in the Golgi lumen as a cis-oriented homodimer, due to strong interactions between coiled-coil regions in the C termini. Dimerization of GREG as well as its Golgi localization depends on a unique tandem repeat sequence within the coiled-coil region. RNA-mediated interference of GREG expression or expression of GREG mutants reveals an essential role for GREG in maintenance of the Golgi integrity. Under these conditions, secretion of the vesicular stomatitis virus glycoprotein protein as a marker for protein transport along the secretory pathway is inhibited, suggesting a loss of Golgi function as well. These results imply the involvement of a luminal protein in Golgi structure and function.

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma.

Results obtained from expression profilings of renal cell carcinoma using different "ome"-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to up-regulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%), and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely three candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain, and ubiquitin carboxyl-terminal hydrolase L1, the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors.

Life-style changes of a halophilic archaeon analyzed by quantitative proteomics.

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.

Proteomic, functional and motif-based analysis of C-terminal Src kinase-interacting proteins.

C-terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine-phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk-interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk-binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk-SH2 domain-binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C-terminus was proved to directly bind to Csk-SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co-localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation-dependent manner and overexpression of Csk, but not its SH2-domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin-Darby canine kidney cells, implying the involvement of Csk-SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.