Showing papers by "Robert Huber published in 2009"

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

Abstract: Prion disease is characterized by the α→β structural conversion of the cellular prion protein (PrPC) into the misfolded and aggregated “scrapie” (PrPSc) isoform. It has been speculated that methionine (Met) oxidation in PrPC may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrPC [rhPrPC(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrPC variant is an α-helix rich protein (like Met-rhPrPC) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrPC variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrPC, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the α→β secondary structure conversion in rhPrPC with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Abstract: Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.

Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3

Abstract: Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal seven-stranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca2+-binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be Ca2+-free and its thermo stability is Ca2+-independent.

The persisting challenge of selective and specific proteasome inhibition.

Abstract: Since the discovery of the proteasome and its structure elucidation intensive research programs in academic institutions and pharmaceutical industries led to identification of a wide spectrum of synthetic and natural small proteasomal inhibitors. Activity studies with these small molecules helped to deeply understand the complex biochemical organization and functioning of the proteasome. The new structural and biochemical insights placed the proteasome as an important anti-cancer drug target, as revealed by the dipeptide boronate proteasome inhibitor, bortezomib, which is currently used for treatment of multiple myeloma. Serious side effects and partial cell resistance against bortezomib demand creation and discovery of new improved generations of more specific and potent proteasomal inhibitors.

Syringolin A Selectively Labels the 20 S Proteasome in Murine EL4 and Wild‐Type and Bortezomib‐Adapted Leukaemic Cell Lines

Abstract: The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite

Abstract: Roundworms of the genus Ascaris are common parasites of the human gastrointestinal tract. A battery of selective inhibitors protects them from host enzymes and the immune system. Here, a metallocarboxypeptidase (MCP) inhibitor, ACI, was identified in protein extracts from Ascaris by intensity-fading MALDI-TOF mass spectrometry. The 67-residue amino acid sequence of ACI showed no significant homology with any known protein. Heterologous overexpression and purification of ACI rendered a functional molecule with nanomolar equilibrium dissociation constants against MCPs, which denoted a preference for digestive and mast cell A/B-type MCPs. Western blotting and immunohistochemistry located ACI in the body wall, intestine, female reproductive tract, and fertilized eggs of Ascaris, in accordance with its target specificity. The crystal structure of the complex of ACI with human carboxypeptidase A1, one of its potential targets in vivo, revealed a protein with a fold consisting of two tandem homologous domains, each containing a β-ribbon and two disulfide bonds. These domains are connected by an α-helical segment and a fifth disulfide bond. Binding and inhibition are exerted by the C-terminal tail, which enters the funnel-like active-site cavity of the enzyme and approaches the catalytic zinc ion. The findings reported provide a basis for the biological function of ACI, which may be essential for parasitic survival during infection.

Pepstatin a derivatives

Abstract: The invention relates to a compound having a structure according to the general formula P3- P2-P1 -P1'-P2' (I), wherein residues P3, P2, P1, P1' and P2' are specifically defined and may be, e. g., certain amino acid residues. The invention further relates to the use of said compound and to a method for synthesizing a peptide.

Dérivés de la pepstatine a

Abstract: La presente invention concerne un compose ayant une structure selon la formule generale P3-P2-P1-P1’-P2’ (I), les residus P3, P2, P1, P1’ et P2’ etant specifiquement definis et pouvant etre, par exemple, certains residus acides amines. L’invention concerne en outre l’utilisation dudit compose et un procede de synthese d’un peptide.