Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24.

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or non-specific binding to unintended membrane protein targets. However, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here, we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored the proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs, but also highlight new approaches for documenting off-target drug binding

Conformational Stability of Hepatitis C Virus NS3 Protease

AbstractThe hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states

Molecular modeling studies suggest that zinc ions inhibit HIV-1 protease by binding at catalytic aspartates.

Human immunodeficiency virus type 1 protease is inhibited in vitro by zinc ions at neutral pH. The binding site of these ions is not known; however, experimental data suggest that binding may occur in the active site. To examine the possibility of zinc binding in the active site, molecular dynamics simulations in the presence and absence of zinc have been carried out to 200 psec. The results are compared with the 2.8-A crystallographic structures of a synthetic HIV-1 protease, and a zinc binding site at the catalytic aspartate residues (Asp-25, Asp-25') is proposed. Molecular dynamics simulations show that the zinc ion remains stably bound in this region, coordinating the carboxylate side chains of both aspartate residues. Interaction with zinc does not disrupt the dimeric structure of the protein or significantly alter the structure of the active site. These data are consistent with experimental studies of HIV-1 protease inhibition by zinc and give strong evidence that this is the binding site that leads to inactivation

Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells

Zinc and Manduca sexta hemocyte functions

Two metalloproteases have recently been linked to the immune response in Lepidoptera. In addition, zinc is highly important in many mammalian immune-related functions. Because of these, we investigated the effect of zinc and two zinc-protease inhibitors on Manduca sexta hemocyte behavior in vitro. Plasmatocytes were significantly more elongated in Grace's medium supplemented with 100 µm zinc chloride than in the absence of zinc. To test whether zinc-dependent proteases were responsible for the increased length seen in the presence of zinc, we tested two zinc-protease inhibitors, phosphoramidon and bestatin. Each resulted in decreased plasmatocyte length compared to the control, but the distributions of lengths differed with each inhibitor. Each inhibitor also affected plasmatocyte network formation in vitro. This work suggests (1) that at least two different zinc proteases are involved in the cellular defense response of M. sexta, and (2) that zinc should be included in media used for in vitro studies of the immune response

Caldolysin, a highly active protease from an extremely Thermophilic Bacterium

Proteases comprise a significant proportion of those proteins which have been subject to detailed characterisation (amino acid sequence and high resolution crystallographic analysis). The extent of research interest in proteolytic enzymes reflects both their historical status, and the practical advantages of proteases as research subjects (available in quantity, extracellular etc.) widely occurring

Die Funktion des Responseregulators ARR2 in der Entwicklung von Arabidopsis thaliana

In dieser Arbeit konnte mittels physiologischer Experimente, Expressionsanalysen, Phosphorylierungsassays und Transaktivierungsanalysen eine komplexe Funktion von ARR2 in verschiedenen Signaltransduktionswegen von Arabidopsis aufgedeckt werden. Die Analyse von Keimlingen in der arr2-Nullmutante zeigt eine hyposensitive Reaktion auf Cytokinin. Mit transienten Transaktivierungsanalysen in Protoplasten konnte die Induktion Cytokinin-responsiver Gene durch ARR2 und ARR2D80E nachgewiesen werden. Zusammenfassend führen diese Ergebnisse zur Hypothese, dass ARR2 eine regulierende Funktion in der Cytokininantwort einnimmt. Die vergleichende physiologische Analyse von Dunkelrotlicht-bestrahlten Keimlingen sowie die Expression des Chalkonsynthasegens identifizieren ARR2 als einen positiven Regulator in der phyA-vermittelten "high irradiance response". Der Nachweis, dass ARR2 in die Ethylensignaltransduktion involviert ist, wurde in verschiedenen Ansätze erbracht. Durch physiologische Analysen unter Verwendung einer arr2-Nullmutante sowie ARR2D80E-überexprimierenden Linien wurde ARR2 als regulierende Komponente der Ethylensignaltransduktion identifiziert. Dies wurde mittels transienter Transaktivierungsanalysen in Arabidopsis-Protoplasten bestätigt. Der zellfreie Phosphorelayassay identifizierte die Ethylenrezeptorhybridkinase ETR1 als putative, stromaufwärtsliegende, phosphorylierende Komponente von ARR2. Die Untersuchungen unter Einsatz der pathogenen Pilze Botrytis cinerea und Peronospora parasitica sowie Trockenstress-Exyperimente lassen eine Rolle von ARR2 in der Pathogen- und dieser abiotischen Stressantwort vermuten. Auf Mikroarrays-basierende Expressionsanalysen der arr2-Nullmutante und ARR2D80E-Pflanzen deuten darauf hin, dass das Zwei-Komponentennetzwerk direkt oder indirekt verschiedene Signaltransduktionswege beeinflussen kann. Zusammenfassend zeigen diese Daten, dass das Zwei-Komponentensystem nicht nur einen primären Signalmechanismus darstellt, sondern zusätzlich das molekulare Grundgerüst für ein komplexes Signaltransduktionsnetzwerk bildet, welches die Feinabstimmung von Signalen übernimmt und die Kommunikation von verschiedenen Signalwegen untereinander ermöglicht

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis