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The Redox Code involves specific, reversible oxidative changes in proteins that modulate protein tertiary structure, interactions, trafficking, and activity, and hence couple the proteome to the metabolic/oxidative state of cells. It is currently a major focus of study in cell biology. Recent studies of dynamic cellular spatial reorganization with MS-based subcellular-spatial-razor proteomics reveal that protein constituents of many subcellular structures, including mitochondria, the endoplasmic reticulum, the plasma membrane, and the extracellular matrix, undergo changes in their subcellular abundance/distribution in response to oxidative stress. These proteins are components of a diverse variety of functional processes spatially distributed across cells. Many of the same proteins are involved in response to suppression of DNA replication indicate that oxidative stress is strongly intertwined with DNA replication/proliferation. Both are replete with networks of moonlighting proteins that show coordinated changes in subcellular location and that include primary protein actuators of the redox code involved in the processing of NAD+ /NADH, NADP+ /NADPH, Cys/CySS, and GSH/GSSG redox couples. Small groups of key proteins such as {KPNA2, KPNB1, PCNA, PTMA, SET} constitute "spatial switches" that modulate many nuclear processes. Much of the functional response involves subcellular protein trafficking, including nuclear import/export processes, vesicle-mediated trafficking, the endoplasmic reticulum/Golgi pathway, chaperone-assisted processes, and other transport systems. This is not visible to measurements of total protein abundance by transcriptomics or proteomics. Comprehensive pictures of cellular function will require collection of data on the subcellular transport and local functions of many moonlighting proteins, especially of those with critical roles in spatial coordination across cells. The proteome-wide analysis of coordinated changes in abundance and trafficking of proteins offered by MS-based proteomics has a unique, crucial role to play in deciphering the complex adaptive systems that underlie cellular function.

Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.

In recent years, large numbers of putative disease biomarkers have been identified. Combinations of protein biomarkers have been proposed to overcome the lack of single, magic-bullet identifiers of disease conditions. The number of biomarkers in a panel must be kept small to avoid the combinatorial explosion that requires very large, uneconomical sample cohorts for validation. Recent results on high sensitivity blood-based diagnostic proteomics (Godovac-Zimmermann, J et al., J. Proteome Res. 2006) suggest that the keys to identifying useful panels include judicious application of physiological knowledge to choose appropriate combinations of local, tissue/disease markers and global, systemic markers and to use very high sensitivity protein detection. Biomarkers that show non-Gaussian landscapes reminiscent of Rene Thom's multiple, stable-state landscapes seem to have the greatest predictive value for breast cancer (Godovac-Zimmermann, J. et al., J. Proteome Res. 2006).

Auoxo3 is a gold(iii) compound endowed with cytotoxic activity towards a variety of malignant cells. Encapsulation of Auoxo3 within horse spleen ferritin (Ft) improves the selectivity of the gold compound towards cancer cells over normal cells. In the current work, the changes in protein expression are presented in response to MCF-7 stimulation with Auoxo3-encapsulated Ft versus the free Au(iii) compound by a label-free proteomics approach. A 159-protein dataset showed significant changes between the stimulations with Auoxo3 and Auoxo3-encapsulated Ft, suggesting that this cellular perturbation caused the alteration of different cellular processes. In detail, roughly 30% of proteins were downregulated mainly in the spliceosome complex (U2AF1, SF3B2, PRPF4, SNSRP200, EFTUD2, PRPF6, and PRPF8) in agreement with the cytostatic effect observed during cellular growth. Another 30% of proteins were upregulated primarily in glutathione biosynthesis, suggesting an alteration of the redox potential, as validated by Western blot analyses. To the best of our knowledge, this work represents the first large scale proteomics study on the effects of a gold-based drug encapsulated within the Ft nanocage on cancer cells.

Background: The purpose of this study is to apply quantitative high‐throughput proteomics methods to investigate dynamic aspects of protein changes in nucleocytoplasmic distribution of proteins and of total protein abundance for MCF‐7 cells exposed to tamoxifen (Tam) in order to reveal the agonistic and antagonistic roles of the drug. / Experimental design: The MS‐based global quantitative proteomics with the analysis of fractions enriched in target subcellular locations is applied to measure the changes in total abundance and in the compartmental abundance/distribution between the nucleus and cytoplasm for several thousand proteins differentially expressed in MCF‐7 cells in response to Tam stimulation. / Results: The response of MCF‐7 cells to the Tam treatment shows significant changes in subcellular abundance rather than in their total abundance. The bioinformatics study reveals the relevance of moonlighting proteins and numerous pathways involved in Tam response of MCF‐7 including some of which may explain the agonistic and antagonistic roles of the drug. / Conclusions: The results indicate possible protective role of Tam against cardiovascular diseases as well as its involvement in G‐protein coupled receptors pathways that enhance breast tissue proliferation.

At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

We have used a proteomics subcellular spatial razor approach to look at changes in total protein abundance and in protein distribution between the nucleus and cytoplasm following exposure of MCF7 breast cancer cells to estradiol. The dominant response of MCF7 cells to estrogen stimulation involves dynamic changes in protein subcellular spatial distribution rather than changes in total protein abundance. Of the 3604 quantitatively monitored proteins, only about 2% show substantial changes in total abundance (>2-fold), whereas about 20% of the proteins show substantial changes in local abundance and/or redistribution of their subcellular location, with up to 16-fold changes in their local concentration in the nucleus or the cytoplasm. We propose that dynamic redistribution of the subcellular location of multiple proteins in response to stimuli is a fundamental characteristic of cells and suggest that perturbation of cellular spatial control may be an important feature of cancer.

Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 (“LDC27”) and 20 residues of D5H (“HKH20”), respectively (Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5H cell binding site, allowed us to isolate a 33-kDa protein (“p33”) that binds specifically and reversibly to H-kininogen with a KD (apparent dissociation constant) of 9 ± 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor (“gC1qR”), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, KD = 240 ± 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5H. Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c- Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.

Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 ("LDC27") and 20 residues of D5H ("HKH20"", respectively (Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5H cell binding site, allowed us to isolate a 33-kDa protein ("p33") that binds specifically and reversibly to H-kininogen with a KD (apparent dissociation constant) of 9 +/- 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor ("gC1qR"), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, KD = 240 +/- 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5H. Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.