Christoph Giese

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF‐7. Of all the tested analogues L‐4‐fluorotryptophan, L‐6‐fluorotryptophan and L‐p‐fluorophenylalanine effectively and irreversibly inhibited cell growth with IC50 values in the low micromolar range (3–15u2005μM). Additionally, using L‐4‐[14C]fluorotryptophan, and L‐6‐[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70‐fold excess of intracellular over extracellular concentrations. We identified systemu2005L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

The Most Stable Protein–Ligand Complex: Applications for One-Step Affinity Purification and Identification of Protein Assemblies†

The purification of protein complexes and large-scale investigations of protein–protein interaction networks have been greatly facilitated through the development of a number of affinity tags such as the cmyc, FLAG, and His6 tags. [2] However, all of the currently available affinity purification systems suffer from dynamic binding equilibria and measurable dissociation rate constants which enable the competitive elution of bound target proteins when an excess of a suitable free ligand is present. This circumstance hampers the quantitative and cost-effective isolation of low-abundance protein complexes. Here, we introduce a new affinity purification system that is derived from type 1 pili of E. coli. Type 1 pili are rigid, filamentous supramolecular protein complexes which are anchored in the cell s outer membrane and extend into the extracellular space. They are composed of four structural protein subunits termed FimH, FimG, FimF, and FimA. In the assembled pilus, these subunits interact noncovalently by a mechanism called donor strand complementation, where the incomplete, immunoglobulin-like fold of one subunit is completed by an N-terminal extension, termed donor strand, of the successive subunit. The complex between FimGt, an N-terminally truncated variant of FimG lacking its own donor strand, and a peptide corresponding to the donor strand of the neighboring subunit FimF (DsF) was found to be the kinetically most stable protein–ligand complex known to date (Figure 1). Here, we establish the FimGt/DsF system for use in the affinity purification of heterooligomeric protein complexes from cell extracts. Utilizing the donor strand of FimF as the affinity tag (termed DsF tag) and FimGt as the binding partner, we demonstrate the one-step purification of DsFtagged E. coli tryptophan synthase, a heterotetrameric abba complex of low cellular abundance. We compare the performance of the DsF tag to that of other commonly used affinity tags and find that, in agreement with the high kinetic stability of the FimGt/DsF complex, enrichment of the tryptophan synthase complex is most efficient for the DsF tag. This result suggests that the DsF tag is most suitable not only for the isolation of low-abundance protein complexes but presumably also for many other technical applications such as, for example, the functional and permanent immobilization of DsF-tagged proteins on surfaces and their detection in cells and on Western blot membranes. As a prerequisite for the technical application of the FimGt/DsF system we first optimized the production of FimGt by producing it in the cytoplasm of E. coli BL21(DE3) cells in the form of inclusion bodies (Figure S1 in the Supporting Information). After solubilization of the inclusion bodies, oxidative refolding, and purification of FimGt by conventional chromatographic techniques, the final yield of purified FimGt was 35 mg per liter of bacterial culture— sufficient amounts for large-scale applications of the FimGt/ DsF system. Quantitative formation of the single, structural disulfide bond was verified by the Ellman assay. The identity of FimGt was confirmed by ESI-MS (expected/measured mass: 13656.9/13657.0 Da). To gain mechanistic insight into the binding reaction between DsF and FimGt, association kinetics were measured for DsF concentrations of 5, 10, 25 and 50 mm while the FimGt concentration was kept constant at 5 mm (Figure 2 a). The reaction rates were dependent on the DsF concentration, indicating that binding of DsF is the rate-limiting step of complex formation. The data were globally fit according to an irreversible second-order reaction and yielded a rate constant of association of (330 8.9)m 1 s . Although relatively slow, the binding of DsF to FimGt is fast enough to allow for technical applications on reasonable time scales. We determined the rate constant for spontaneous dissociation/unfolding of the FimGt/DsF complex at pH 8.0 and 25 8C to be Figure 1. Crystal structure of the FimGt/DsF complex (3BFQ.pdb). FimGt is shown as a gray surface, the DsF peptide as stick representation. Residues of DsF that point towards FimGt are in bold and their position in the structure is indicated by arrows.

The Cryoelectron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod

Summary Adhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro.

Treating insect-bite hypersensitivity in horses with active vaccination against IL-5

Background Insect‐bite hypersensitivity is the most common allergic dermatitis in horses. Excoriated skin lesions are typical symptoms of this seasonal and refractory chronic disease. On a cellular level, the skin lesions are characterized by massive eosinophil infiltration caused by an underlying allergic response. Objective To target these cells and treat disease, we developed a therapeutic vaccine against equine IL‐5 (eIL‐5), the master regulator of eosinophils. Methods The vaccine consisted of eIL‐5 covalently linked to a virus‐like particle derived from cucumber mosaic virus containing the tetanus toxoid universal T‐cell epitope tt830‐843 (CMVTT). Thirty‐four Icelandic horses were recruited and immunized with 400 &mgr;g of eIL‐5–CMVTT formulated in PBS without adjuvant (19 horses) or PBS alone (15 horses). Results The vaccine was well tolerated and did not reveal any safety concerns but was able to induce anti–eIL‐5 autoantibody titers in 17 of 19 horses. This resulted in a statistically significant reduction in clinical lesion scores when compared with previous season levels, as well as levels in placebo‐treated horses. Protection required a minimal threshold of anti–eIL‐5 antibodies. Clinical improvement by disease scoring showed that 47% and 21% of vaccinated horses reached 50% and 75% improvement, respectively. In the placebo group no horse reached 75% improvement, and only 13% reached 50% improvement. Conclusion Our therapeutic vaccine inducing autoantibodies against self IL‐5 brings biologics to horses, is the first successful immunotherapeutic approach targeting a chronic disease in horses, and might facilitate development of a similar vaccine against IL‐5 in human subjects. Graphical abstract Figure. No Caption available.

Accelerating the Association of the Most Stable Protein–Ligand Complex by more than Two Orders of Magnitude

The complex between the bacterial typeu20051 pilus subunit FimG and the peptide corresponding to the N-terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (KD ) of 1.5×10(-20) u2009m. However, the complex only exhibits a slow association rate of 330u2009m(-1) u2009s(-1) that limits technical applications, such as its use in affinity purification. Herein, a structure-based approach was used to design pairs of FimGt (a FimG variant lacking its own N-terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A KD u2005value of 8.8×10(-22) u2009m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.

Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms

Cdc48 is a AAA+ ATPase that plays an essential role for many cellular processes in eukaryotic cells. An archaeal homologue of this highly conserved enzyme was shown to directly interact with the 20S proteasome. Here, we analyze the occurrence and phylogeny of a Cdc48 homologue in Actinobacteria and assess its cellular function and possible interaction with the bacterial proteasome. Our data demonstrate that Cdc48-like protein of actinobacteria (Cpa) forms hexameric rings and that the oligomeric state correlates directly with the ATPase activity. Furthermore, we show that the assembled Cpa rings can physically interact with the 20S core particle. Comparison of the Mycobacterium smegmatis wild-type with a cpa knockout strain under carbon starvation uncovers significant changes in the levels of around 500 proteins. Pathway mapping of the observed pattern of changes identifies ribosomal proteins as a particular hotspot, pointing amongst others toward a role of Cpa in ribosome adaptation during starvation.

Struktur, Assemblierung und Stabilität von Typ-1-Pili

Type 1 pili are extracellular, supramolecular protein complexes required for the attachment of pathogenic E. coli strains to host cells. Hundreds to thousands of protein subunits are assembled within minutes in vivo and form filaments with unique kinetic stability against dissociation. Here, we review recent work on the structure, assembly mechanism and potential technical applications derived from this exciting biological system.