Design of hairpin ribozyme variants with improved activity for poorly processed substrates.

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y⁻² N⁻¹ *G+¹ U+² Y+³ B+⁴, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U⁻² G⁻¹ *G+¹ U+² A+³ A+⁴ sites.

Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae.

The trypanosomatid protozoon Leishmania tarentolae is a well-established model organism for studying causative agents of several tropical diseases that was more recently developed as a host for recombinant protein production. Although several expression architectures based on foreign RNA polymerases have been established for this organism, all of them rely on integration of the expression cassette into the genome. Here, we exploit a new type of expression architecture based on linear elements.

Spatial cycles in G-protein crowd control.

The nature of living systems and their apparent resilience to the second law of thermodynamics has been the subject of extensive investigation and imaginative speculation. The segregation and compartmentalization of proteins is one manifestation of this departure from equilibrium conditions; the effect of which is now beginning to be elucidated. This should not come as a surprise, as even a cursory inspection of cellular processes reveals the large amount of energetic cost borne to maintain cell-scale patterns, separations and gradients of molecules.

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae.

The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre.

In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.

Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ.

Small-molecule inhibition of APT1 affects Ras localization and signaling.

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells.

A new model for the transition of APAF-1 from inactive monomer to caspase-activating apoptosome.

The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro.

A supramolecular polymer as a self-assembling polyvalent scaffold.

Binding bacteria: Discotic molecules self-assemble into columnar supramolecular polymers that show strong polyvalent binding to bacteria by virtue of mannose ligands attached at their periphery (orange; see picture). The reversible formation of the supramolecular polymers allows simple mixing of differently substituted monomers and the optimization of bacterial aggregation.

Reassessment of the role of FKBP38 in the Rheb/mTORC1 pathway.

The small G-protein Rheb regulates cell growth via the mTORC1 complex by incompletely understood mechanisms. Recent studies document that Rheb activates mTORC1 via direct, GTP-dependent interaction with the peptidyl-prolyl-cis/trans-isomerase FKBP38, which is proposed to act as an inhibitor of mTORC1. We have conducted a comprehensive biochemical characterization of the Rheb/FKBP38 interaction.

Development of tau aggregation inhibitors for Alzheimer's disease.

A variety of human diseases are suspected to be directly linked to protein misfolding. Highly organized protein aggregates, called amyloid fibrils, and aggregation intermediates are observed; these are considered to be mediators of cellular toxicity and thus attract a great deal of attention from investigators. Neurodegenerative pathologies such as Alzheimer's disease account for a major part of these protein misfolding diseases.

Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli.

The guanine nucleotide binding protein Rab8A controls the final steps of exocytosis in mammalian cells. It has been implicated in the regulation of apical protein localization in intestinal epithelial cells and ciliary biogenesis. The in vitro structural and biochemical characterization of Rab8A and its interaction with regulator and effector molecules has been hampered by its insolubility in Escherichia coli expression systems.

Biophysical analysis of the interaction of Rab6a GTPase with its effector domains.

Rab GTPases are key regulators of intracellular vesicular transport that control vesicle budding, cargo sorting, transport, tethering, and fusion. In the inactive (GDP-bound) conformation, Rab GTPases are targeted to intracellular compartments where they are converted into the active GTP-bound form and recruit effector domain containing proteins. Rab6a has been implicated in dynein-mediated vesicle movement at the Golgi apparatus and shown to interact with a plethora of effector proteins.

Specificity of interactions between mDia isoforms and Rho proteins.

Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily.

Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation.

Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase.

Miniaturization of primary porcine urinary bladder epithelial cell cultures and application for gene expression studies after exposure to benzo[a]pyrene.

Benzo[a]pyrene (BaP) is an environmental pollutant used as a key marker substance for polycyclic aromatic hydrocarbons (PAHs). PAHs are believed to play a prominent role in the development of bladder cancer. A test system based on primary porcine urinary bladder epithelial cells (PUBEC) has been utilized as an in vitro model for urinary bladder epithelium.

A structural model of the GDP dissociation inhibitor rab membrane extraction mechanism.

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.

A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.

Sorting nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX-BAR domain protein.

The PX-BAR membrane-remodeling unit of sorting nexin 9.

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro.

The epsilon-isoform of PKC mediates the hypertonic activation of cation channels in confluent monolayers of rat hepatocytes.

We were interested whether PKC alpha, delta, epsilon or zeta is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKC alpha, delta and epsilon were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKC delta and epsilon did respond to hypertonic stress. Furthermore, chelation of intracellular Ca(++) by BAPTA-AM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKC delta and epsilon) activated HICC currents under isotonic conditions.

Construction and analysis of Leishmania tarentolae transgenic strains free of selection markers.

The trypanosomatid protozoan Leishmania tarentolae has been extensively used as a model system for studying causative agents of several tropical diseases and more recently as a host for recombinant protein production. Here we analyze the rates of partial or complete deletions of expression cassettes integrated into small ribosomal RNA and tubulin gene clusters as well as into ornithine decarboxylase gene of L. tarentolae.

Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators.

Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab escort protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated proteins is complicated by their low solubility in aqueous solutions.

The protein truncation test in mutation detection and molecular diagnosis.

The protein truncation test (PTT) is a simple and fast method to screen for biologically relevant gene mutations. The method is based on the size analysis of products resulting from in vitro transcription and translation. Proteins of lower mass than the expected full-length protein represent translation products derived from truncating frame shift or stop mutations in the analyzed gene.

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells.

Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography-mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells.

A size filtration approach to purify low affinity complexes for crystallization.

Low affinity protein complexes are difficult to isolate and handle in crystallization experiments. Size-exclusion chromatography often does not allow purification of the homogeneous complex. Here we used a size-filtration approach for the purification and concentration of the 19 microM affinity complex of yeast Rab-GTPase and its guanine nucleotide disassociation inhibitor (GDI).