Heterologe Expression, Kristallisation und Untersuchungen zur Struktur von Bos taurus β\beta-Arrestin und Rattus norvegicus PAR-4

$\beta$-$\textbf{arrestins}$ are essential regulators of G-Protein coupled receptors. In addition to terminatingthe signal cascade, they mediate the endocytosis of the receptors through "clathrin coatedpits." In this work the protocols for the heterologous expression and purification of bovine$\beta$-arrestin in $\textit{S.cerevisiae}$ are established. Through mutagenesis affinity tags were added to the$\beta$-arrestin gene. Affinity chromatography yielded up to 2.7 mg of protein per gram of cells.The purity of the protein was optimized by usage of two different affinity tags (C- and Nterminus)and sequential purification. The stability against oxidation and the solubility of theprotein were significantly increased through the mutagenesis of four cysteine residues toserines and the employment of high concentrations of phosphate.The functionality of the heterologously expressed $\beta$-arrestin was demonstrated by means of itscapacity to bind rhodopsin. Using tartrate as a precipitant, crystals of purified protein weregrown as large as 800 pm; however the low stability and poor diffraction hamperedcrystallographic success. By co-crystallization of $\beta$-arrestin with its ligand inositolhexaphosphate(IP$_{6}$) the diffraction quality of the crystals was dramatically improved. Neitherthe type nor the position of the affinity tag used had any significant influence on theappearance of the crystals hence on the packing of the molecules. Crystallographic data setsobtained using synchrotron radiation are currently being analyzed to solve the three-dimensionalstructure of $\beta$-arrestin by molecular replacement of the known structure of visualarrestin. Furthermore co-crystallized heavy metal derivatives of $\beta$-arrestin were performed,thereby enabling structure-determination independent of the arrestin-model by means ofisomorphous replacement.A first result to emerge from this analysis was the mapping of the IP$_{6}$-binding site to the C-terminaldome of the $\beta$-arrestin molecule as previously proposed. Furthermore there was noevidence in support of additional binding-sites for IP$_{6}$ as postulated in the literature.Further work will involve the improvement of the diffraction quality of the crystals,the determination of the three-dimensional structure of $\beta$-arrestin as well as attempts toco-crystallize $\beta$-arrestin together with other proteins which interact specifically with it.$\textbf{PAR-4}$ plays an essential role mediating the susceptibility of cells towards apoptosis, is activein certain tumors and is a protagonist responsible for some symptoms of variousneurodegenerative diseases such as the Alzheimer's or Parkinson's. In the apoptotic signalcascade PAR-4 seems to act at an early stage.With the experiments presented in this thesis attempts were made to heterologously expressand to crystallize PAR-4. First trials failed due to the low solubility of the protein, a problemwhich was overcome with high concentrations of divalent cations. Using affinity tags andchromatographic methods sufficient amount of highly purified protein, suitable forcrystallization were obtained from E.coli (10 mg/g cells) as well as from $\textit{S.cerevisiae}$ (2 mg/gcells). Functionality of the protein on the physiological level was tested with a kinase assay.The structure of the isolated protein and its integrity was investigated and preliminarilydescribed by biophysical techniques such as NMR and FTIR-spectroscopy. Crystallizationattempt so far have yielded crystals unsuitable for crystallographic pursuits. These resultsserve as groundwork towards the three dimensional structure of PAR-4

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteins via its C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to be P41212. The unit-cell parameters are a = b = 115.351, c = 123.663 Å, α = β = γ = 90°

A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate.

The dynamic modification of proteins with ubiquitin is a key regulation paradigm in eukaryotic cells that controls stability, localization, and function of the vast majority of intracellular proteins. Here we describe a robust fluorescence intensity assay for monitoring the enzymatic activity of deubiquitinating proteases, which reverse ubiquitin modifications and comprise over 100 members in humans. The assay was developed for the catalytic domain of human ubiquitin-specific protease 2 (USP2) and human ubiquitin carboxyterminal hydrolase L3 (UCH-L3), and makes use of the novel substrate ubiquitin-rhodamine110-glycine. The latter combines the advantages of a high dynamic range and beneficial optical properties. Its enzymatic behavior is characterized by the kinetic constants K(m)=1.5 microM, k(cat) = 0.53s(-1) and k(cat)/K(m) = 3.5 x 10(5)M(-1) s(-1) for USP2 and K(m) = 34 nM, k(cat)=4.72s(-1), and k(cat)/K(m) = 1.4 x 10(8)M(-1) s(-1) for UCH-L3. This new assay is suitable for inhibitor screening and characterizations, and has been established for the 384-well plate format using protease concentrations of 120 pM for USP2 and 1 pM for UCH-L3 and substrate concentrations of 100 nM for both enzymes. Due to the low protease concentrations and high sensitivity, this assay would allow the determination of inhibitory constants in the subnanomolar range

Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor.

BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2.

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay