A chemical compound inhibiting the Aha1-Hsp90 chaperone complex.

The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target.

Inhibitors of macrophage infectivity potentiator-like PPIases affect neisserial and chlamydial pathogenicity.

The pathogenic bacteria Chlamydia trachomatis, Neisseria gonorrhoeae and Neisseria meningitidis express the surface-exposed macrophage infectivity potentiator (MIP)-like protein, which plays a role in their pathogenicity. MIP exhibits a peptidyl-prolyl isomerase (PPIase) activity that is inhibited by rapamycin and FK506. In this study, pipecolic acid derivatives were tested for their activity against the chlamydial and neisserial MIP.

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear.

The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.

Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix.

Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly.

Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L.

Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.

The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle.

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.

Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature.

ERp29 deficiency affects sensitivity to apoptosis via impairment of the ATF6-CHOP pathway of stress response.

Endoplasmic reticulum protein 29 (ERp29) belongs to the redox-inactive PDI-Dβ-subfamily of PDI-proteins. ERp29 is expressed in all mammalian tissues examined. Especially high levels of expression were observed in secretory tissues and in some tumors.

The FKBP-type domain of the human aryl hydrocarbon receptor-interacting protein reveals an unusual Hsp90 interaction.

The aryl hydrocarbon receptor-interacting protein (AIP) has been predicted to consist of an N-terminal FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal tetratricopeptide repeat (TPR) domain, as typically found in FK506-binding immunophilins. AIP, however, exhibited no inherent FK506 binding or PPIase activity. Alignment with the prototypic FKBP12 showed a high sequence homology but indicated inconsistencies with regard to the secondary structure prediction derived from chemical shift analysis of AIP(2-166).

PPARα modulates the TSH β-subunit mRNA expression in thyrotrope TαT1 cells and in a mouse model.

Scope: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis.

Methods And Results: Thyroid-stimulating hormone β-subunit (TSHβ) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.

¹H, ¹³C and ¹⁵N resonance assignments of human parvulin 17.

A 25-residue elongation at the N-terminus endows parvulin 17 (Par17) with altered functional properties compared to parvulin 14 (Par14), such as an enhanced influence on microtubule assembly. Therefore the three-dimensional structure of this N-terminal elongation is of particular interest. Here, we report the nearly complete (1)H, (13)C and (15)N chemical shift assignments of Par17.

Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation.

Polar transport of the plant hormone auxin is controlled by PIN- and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1.

NMR assignments of the FKBP-type PPIase domain of the human aryl-hydrocarbon receptor-interacting protein (AIP).

The aryl-hydrocarbon receptor-interacting protein (AIP) interacts with several protein binding partners and has been associated with pituitary tumor development. Here, we report nearly complete (1)H, (13)C and (15)N chemical shift assignments for the N-terminal AIP(2-166) segment, which has been predicted to represent a FKBP-type PPIase domain. Sequence alignment with the prototypic FKBP12, however, reveals disagreements between the AIP chemical shift index consensus and the corresponding FKBP12 secondary structure elements.

NMR assignments of the FKBP-type PPIase domain of FKBP42 from Arabidopsis thaliana.

The Atfkbp42 gene is associated with reduced and disoriented growth of Arabidopsis thaliana. Resonance assignments are reported for the FKBP-type PPIase domain of AtFKBP42. Signal intensities reveal an additional structure element that is atypical for such FKBP domains.

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration.

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1.

Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity.

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay.

Calcineurin inhibitors: status quo and perspectives.

Despite the fact that cyclosporin A (CsA) and tacrolimus (FK506) are very potent drugs in the treatment of serious autoimmune diseases and in the prevention of graft vs. host reactions or tissue rejections after allo- or xenotransplantations, modern transplantation medicine attempts to develop alternative medication regimes without these calcineurin inhibitors. The primary motivation for this endeavor is the high incidence of dramatic side effects upon immunosuppressive therapy.

Pipecolic acid derivatives as small-molecule inhibitors of the Legionella MIP protein.

The macrophage infectivity potentiator (MIP) protein is a major virulence factor of Legionella pneumophila, the causative agent of Legionnaires' disease. MIP belongs to the FK506-binding proteins (FKBP) and is necessary for optimal intracellular survival and lung tissue dissemination of L. pneumophila.

High density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates.

Protein function is highly regulated in pathways that are responsible for complex biochemical mechanisms such as growth, metabolism, and signal transduction. One of the most important mechanisms is posttranslational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation, glycosylation, and sumoylation resulting in a more than 100-fold higher complexity (Geiss-Friedlander and Melchior, Nat Rev Mol Cell Biol 8, 947-956, 2007; Hunter, Mol Cell 28, 730-738, 2007). This chapter presents a very efficient way to detect potential phosphorylation sites in protein families using overlapping peptides covering the complete primary structures (peptide scans) immobilized on glass slides.

The novel calcineurin inhibitor CN585 has potent immunosuppressive properties in stimulated human T cells.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library.

High-density peptide microarrays for reliable identification of phosphorylation sites and upstream kinases.

The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity.

Identification of a disulfide bridge essential for transport function of the human proton-coupled amino acid transporter hPAT1.

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown.