Impact of left ventricular assist device therapy on the cardiac proteome and metabolome composition in ischemic cardiomyopathy.

The changes in the myocardial proteome and metabolome associated with left ventricular assist device (LVAD) therapy in patients with ischemic cardiomyopathy (ICM) are poorly characterized. We investigated the impact of mechanical unloading following LVAD therapy on the myocardial proteome and metabolome. Matched samples of 5 patients' myocardial tissue, harvested at the time of LVAD implant ("pre-LVAD") or heart transplant ("post-LVAD"), were studied by quantitative proteomics and metabolomics as well as being probed for T-tubule structure and connexin-43 distribution. Moreover, pre-LVAD proteome profiles of ICM context were bioinformatically compared to pre-LVAD proteome profiles of dilated cardiac myopathy (DCM). More than 2120 proteins were reliably identified and quantified in paired patient samples. LVAD therapy led to proteomic remodeling, including reduced levels of α-1-antichymotrypsin together with an overall decrease of immune response proteins and an increase of proteins involved in membrane biology. Metabolomics highlighted increased glucose and glucose-6-phosphate levels in the left ventricle upon LVAD therapy. Wheat germ agglutinin staining demonstrated improved T-tubule structure. Connexin-43 displayed a trend for more pronounced intercalated disc localization. In comparing pre-LVAD proteome profiles of ICM context with pre-LVAD proteome profiles of dilated cardiac myopathy (DCM), we noticed an overrepresentation in ICM of proteins associated with humoral immune response. Our findings underline an impact of LVAD therapy on left ventricular biology in ICM. The proteomic, metabolomic, and structural alterations described here are typically associated with cardiac recovery. On the molecular level, our findings indicate the possibility of cardiac remodeling under LVAD therapy in ICM.

Specificity profiling of human trypsin-isoenzymes.

In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1' position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1' and P2' preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2' is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.

The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B.

The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations.

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

Enrichment of protein N-termini by charge reversal of internal peptides.

Protein N-termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N-terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N-terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N-terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof-of-concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N-termini. We further show the application of this strategy in investigating the N-terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel-based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products.

A novel role for inhibitor of apoptosis (IAP) proteins as regulators of endothelial barrier function by mediating RhoA activation.

Inhibitor of apoptosis (IAP) proteins, such as XIAP or cIAP1/2, are important regulators of apoptosis in cancer cells, and IAP antagonists are currently evaluated as antitumor agents. Beyond their function in cancer cells, this study demonstrates a novel role of IAPs as regulators of vascular endothelial permeability. Two structurally different IAP antagonists, ABT and Smac085, as well as silencing of IAPs, reduced the thrombin receptor-activating peptide (TRAP)-induced barrier dysfunction in human endothelial cells as assessed by measuring macromolecular permeability or transendothelial electrical resistance. ABT diminished thrombin-evoked stress fiber formation, activation of myosin light chain 2, and disassembly of adherens junctions independent of calcium signaling, protein kinase C, and mitogen-activated protein kinases. Interestingly, ABT and silencing of IAPs, in particular XIAP, reduced the TRAP-evoked RhoA activation, whereas Rac1 was not affected. XIAP and, to a lesser extent, cIAP1 were found to directly interact with RhoA independently of the RhoA activation status. Under cell-free conditions, XIAP did not induce an ubiquitination of RhoA. In summary, our work discloses IAPs as crucial regulators of endothelial permeability and suggests IAP inhibition as interesting approach for the prevention of endothelial barrier dysfunction.

Automated peptide mapping and protein-topographical annotation of proteomics data.

BACKGROUND: In quantitative proteomics, peptide mapping is a valuable approach to combine positional quantitative information with topographical and domain information of proteins. Quantitative proteomic analysis of cell surface shedding is an exemplary application area of this approach. RESULTS: We developed ImproViser ( http://www.improviser.uni-freiburg.de) for fully automated peptide mapping of quantitative proteomics data in the protXML data. The tool generates sortable and graphically annotated output, which can be easily shared with further users. As an exemplary application, we show its usage in the proteomic analysis of regulated intramembrane proteolysis. CONCLUSION: ImproViser is the first tool to enable automated peptide mapping of the widely-used protXML format.

Flavopiridol protects against inflammation by attenuating leukocyte-endothelial interaction via inhibition of cyclin-dependent kinase 9.

OBJECTIVE: The cyclin-dependent kinase (CDK) inhibitor flavopiridol is currently being tested in clinical trials as anticancer drug. Beyond its cell death-inducing action, we hypothesized that flavopiridol affects inflammatory processes. Therefore, we elucidated the action of flavopiridol on leukocyte-endothelial cell interaction and endothelial activation in vivo and in vitro and studied the underlying molecular mechanisms. METHODS AND RESULTS: Flavopiridol suppressed concanavalin A-induced hepatitis and neutrophil infiltration into liver tissue. Flavopiridol also inhibited tumor necrosis factor-α-induced leukocyte-endothelial cell interaction in the mouse cremaster muscle. Endothelial cells were found to be the major target of flavopiridol, which blocked the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin), as well as NF-κB-dependent transcription. Flavopiridol did not affect inhibitor of κB (IκB) kinase, the degradation and phosphorylation of IκBα, nuclear translocation of p65, or nuclear factor-κB (NF-κB) DNA-binding activity. By performing a cellular kinome array and a kinase activity panel, we found LIM domain kinase-1 (LIMK1), casein kinase 2, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), CDK4, CDK6, CDK8, and CDK9 to be influenced by flavopiridol. Using specific inhibitors, as well as RNA interference (RNAi), we revealed that only CDK9 is responsible for the action of flavopiridol. CONCLUSIONS: Our study highlights flavopiridol as a promising antiinflammatory compound and inhibition of CDK9 as a novel approach for the treatment of inflammation-associated diseases.

Inhibitor of apoptosis proteins as novel targets in inflammatory processes.

OBJECTIVE: Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NFκB signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. METHODS AND RESULTS: In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNFα-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte-endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NFκB signaling but decreased the TNFα-induced activation of the TGF-ß-activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. CONCLUSIONS: Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells.

A human endogenous retrovirus encoded protease potentially cleaves numerous cellular proteins.

BACKGROUND: A considerable portion of the human genome derives from retroviruses inherited over millions of years. Human endogenous retroviruses (HERVs) are usually severely mutated, yet some coding-competent HERVs exist. The HERV-K(HML-2) group includes evolutionarily young proviruses that encode typical retroviral proteins. HERV-K(HML-2) has been implicated in various human diseases because transcription is often upregulated and some of its encoded proteins are known to affect cell biology. HERV-K(HML-2) Protease (Pro) has received little attention so far, although it is expressed in some disease contexts and other retroviral proteases are known to process cellular proteins. RESULTS: We set out to identify human cellular proteins that are substrates of HERV-K(HML-2) Pro employing a modified Terminal Amine Isotopic Labeling of Substrates (TAILS) procedure. Thousands of human proteins were identified by this assay as significantly processed by HERV-K(HML-2) Pro at both acidic and neutral pH. We confirmed cleavage of a majority of selected human proteins in vitro and in co-expression experiments in vivo. Sizes of processing products observed for some of the tested proteins coincided with product sizes predicted by TAILS. Processed proteins locate to various cellular compartments and participate in diverse, often disease-relevant cellular processes. A limited number of HERV-K(HML-2) reference and non-reference loci appears capable of encoding active Pro. CONCLUSIONS: Our findings from an approach combining TAILS with experimental verification of candidate proteins in vitro and in cultured cells suggest that hundreds of cellular proteins are potential substrates of HERV-K(HML-2) Pro. It is therefore conceivable that even low-level expression of HERV-K(HML-2) Pro affects levels of a diverse array of proteins and thus has a functional impact on cell biology and possible relevance for human diseases. Further studies are indicated to elucidate effects of HERV-K(HML-2) Pro expression regarding human substrate proteins, cell biology, and disease. The latter also calls for studies on expression of specific HERV-K(HML-2) loci capable of encoding active Pro. Endogenous retrovirus-encoded Pro activity may also be relevant for disease development in species other than human.

Atrial natriuretic peptide protects against histamine-induced endothelial barrier dysfunction in vivo.

Endothelial barrier dysfunction is a hallmark of many severe pathologies, including sepsis or atherosclerosis. The cardiovascular hormone atrial natriuretic peptide (ANP) has increasingly been suggested to counteract endothelial leakage. Surprisingly, the precise in vivo relevance of these observations has never been evaluated. Thus, we aimed to clarify this issue and, moreover, to identify the permeability-controlling subcellular systems that are targeted by ANP. Histamine was used as important pro-inflammatory, permeability-increasing stimulus. Measurements of fluorescein isothiocyanate (FITC)-dextran extravasation from venules of the mouse cremaster muscle and rat hematocrit values were performed to judge changes of endothelial permeability in vivo. It is noteworthy that ANP strongly reduced the histamine-evoked endothelial barrier dysfunction in vivo. In vitro, ANP blocked the breakdown of transendothelial electrical resistance (TEER) induced by histamine. Moreover, as judged by immunocytochemistry and Western blot analysis, ANP inhibited changes of vascular endothelial (VE)-cadherin, beta-catenin, and p120(ctn) morphology; VE-cadherin and myosin light chain 2 (MLC2) phosphorylation; and F-actin stress fiber formation. These changes seem to be predominantly mediated by the natriuretic peptide receptor (NPR)-A, but not by NPR-C. In summary, we revealed ANP as a potent endothelial barrier protecting agent in vivo and identified adherens junctions and the contractile apparatus as subcellular systems targeted by ANP. Thus, our study highlights ANP as an interesting pharmacological compound opening new therapeutic options for preventing endothelial leakage.

Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics.

Biochemical profiling of active site specificity is a crucial step to characterize proteases, which play key roles in health and disease. Here, we present a protocol using proteome-derived peptide libraries in combination with quantitative proteomics to simultaneously identify cleavage motifs N- and C-terminal to the scissile peptide bond. First, bacterial or eukaryotic cell lysate is used to generate peptide libraries. Without further chemical modification, peptide libraries are then split into control and treated (incubate with active protease) aliquots. Control and treated libraries are stable isotope-labeled, mixed, and analyzed by liquid chromatography-tandem mass spectrometry. Enriched, semi-specific peptides represent the cleavage products of the test protease and the entire peptide sequence that encompasses the scissile peptide bond is reconstructed bioinformatically. The method is fast, cost-effective, and suited for proteases with narrow or loose specificity.

Proteomics highlights decrease of matricellular proteins in left ventricular assist device therapy†.

OBJECTIVES: We investigated the impact of mechanical unloading with a left ventricular assist device (LVAD) on the myocardial proteome. METHODS: We collected 11 patient-matched samples of myocardial left ventricular tissue of patients with non-ischaemic dilate cardiomyopathy, harvested at time of LVAD implant ('pre-LVAD') and heart transplant ('post-LVAD'). Samples were studied by quantitative proteomics. Further we performed histological assessment of deposited collagens and immune infiltration in both pre- and post-LVAD samples. RESULTS: A core set of >1700 proteins was identified and quantified at a false discovery rate <1%. The previously established decrease post-LVAD of alpha-1-antichymotrypsin was corroborated. We noted a post-LVAD decrease of matricellular proteins and proteoglycans such as periostin and versican. Also, proteins of the complement system and precursors of cardiac peptide hormones were decreased post-LVAD. An increase post-LVAD was evident for individual proteins linked to the innate immune response, proteins involved in diverse metabolic pathways, and proteins involved in protein synthesis. Histological analysis did not reveal significant alterations post-LVAD of deposited collagens or immune infiltration. The proteomic data further highlighted a pronounced inter-patient heterogeneity with regards to the impact of LVAD therapy on the left ventricular myocardial proteome. Finally, the proteomic data showed differential proteolytic processing in response to LVAD therapy. CONCLUSIONS: Our findings underline a strong impact of LVAD therapy on the left ventricular myocardial proteome. Together with previous studies, protein markers of LVAD therapy such as alpha-1-antichymotrypsin are becoming apparent. Further, matricellular proteins are emerging as important components in response to LVAD therapy.

Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay.

The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer's tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell(®) tracer assays and two commercial impedance devices (xCELLigence(®), ECIS(®)). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments.

Pregnancy Specific ß-1 Glycoprotein 1 is Expressed in Pancreatic Ductal Adenocarcinoma and its Subcellular Localization Correlates with Overall Survival.

Proteins of the pregnancy specific ß-1 glycoprotein (PSG) family are renowned for their elevated expression during pregnancy. Only few reports have investigated their expression in adenocarcinomas. We studied the expression of PSG1 in pancreatic adenocarcinoma (PDAC). In a cohort of 104 patient samples, immunohistochemical analysis determined PSG1 expression in every specimen. PSG1 was found at apical and cytoplasmic localization or solely at cytoplasmic localization, with the latter case being correlated to shortened median survival (25 vs 11 months, logrank p-value < 0.001). At the same time, enzyme linked immunosorbent assay (ELISA) did not detect elevated PSG1 levels in the plasma of PDAC patients as opposed to the plasma of healthy, non-pregnant control individuals. We also probed the impact of PSG1 expression in a murine tumor model system, using subcutaneous injection of Colo-26 cells into immunocompetent BALB/c mice. Here, tumor growth was not affected by the expression of human PSG1. Our study reaffirms interest into the tumor-contextual biology of PSG proteins.

Impact of routinely employed procedures for tissue processing on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

PURPOSE: FFPE (formalin fixed, paraffin embedded) tissue cohorts represent an enduring archive of clinical specimens. Proteomic analysis of FFPE tissues is gaining interest for the in-depth analysis of aberrant proteome composition. Procedures for FFPE tissue processing are standardized but there is diversity regarding the different processing systems. This work focuses on three different processing methods commonly used in large European pathology institutes. EXPERIMENTAL DESIGN: Formalin fixed tissue specimens of different tumors were serially sliced and processed with three different processing systems (xylene, ethanol/vacuum or microwave based). After paraffin embedding, they were subjected to MS-based proteomic analysis to investigate the impact of tissue processing techniques on the quality of proteomic analysis. Results were compared with proteomic analysis of corresponding cryopreserved tissue specimens. RESULTS: All processing techniques achieved very good proteome coverage similar to the cryopreserved counterpart. Gene ontology profiles, relative protein abundances, and peptide modifications such as methionine oxidation or proteolytic truncation were highly similar for all techniques as well as for the cryopreserved samples. CONCLUSIONS AND CLINICAL RELEVANCE: The results show that different processing procedures do not impede proteomic analysis as a robust and powerful approach for the identification of protein determinants and markers of disease processes and highlights the general robustness of FFPE-tissue based proteomics.

Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9.

BACKGROUND AND PURPOSE: Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation. EXPERIMENTAL APPROACH: Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine. KEY RESULTS: In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine. CONCLUSIONS AND IMPLICATIONS: Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound.

The universal stress protein UspC scaffolds the KdpD/KdpE signaling cascade of Escherichia coli under salt stress.

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K(+)-transport system KdpFABC in response to K(+) limitation or salt stress. Under K(+) limiting conditions the Kdp system restores the intracellular K(+) concentration, while in response to salt stress K(+) is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K(+), so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD-->KdpE-->DNA) resulting in phosphorylation of KdpE at a K(+) concentration that would otherwise almost prevent phosphorylation. In agreement, in a DeltauspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K(+) limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE~P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.