Mutants of human O6-alkylguanine-DNA alkyltransferase with improved properties

The invention relates to human O6-alkylguanine-DNA alkyltransferase (hAGT) mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localization of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as sol. protein and improved stability in various hosts; (d) improved stability under oxidizing conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro soly.; (h) improved reactivity against O6-alkylguanine substrates; (i) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted 06-alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally 1 to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest. [on SciFinder (R)]

Mutants of o6-alkylguanine-dna alkyltransferase

Christopher Chidley, Thomas Gronemeyer, Christian Heinis, Kai Johnsson, Alexandre Juillerat, Antje Keppler

The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus (c) improved expression yield as soluble protein and improved stability in various hosts (d) improved stability under oxidising conditions (e) improved stability within cells after reaction with a substrate (f) improved stability outside cells before and after reaction with a substrate (g) improved in vitro solubility (h) improved reactivity against 0-alkylguanine substrates (1) reduced reactivity against DNA-based substrates and (j) reduced reactivity against N-substituted 0-alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally I to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest.

2006

Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

Thomas Gronemeyer, Kai Johnsson, Alexandre Juillerat, Antje Keppler, Horst Pick, Horst Vogel

We report here the generation of mutants of the human O6-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter mols. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O6-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O6-benzylguanine derivs., the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small mols. and therefore should become important tools for studying protein function in living cells. In addn. to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O6-benzylguanine. [on SciFinder (R)]

2003

Innate Barriers to Genome Invasion

Pierre Maillard

Over the course of evolution, retroviruses and their hosts have been in constant conflict, resulting in a long-term evolutionary "arms race" in which host and pathogen alternately gain selective advantages through functional innovations. On the host side, cells of higher eukaryotes have evolved to encode numerous inhibitory activities called restriction factors to block retroviral replication. Conversely, retroviruses have evolved to exploit numerous host cellular components to replicate and to efficiently counter host intrinsic lines of defence. The current acquired immunodeficiency syndrome (AIDS) pandemic exemplifies this struggle, as its causative agent the human immunodeficiency virus (HIV) was found to overcome several host-encoded restriction factors active against other retroviruses. TRIM5α is one such restriction factor, which inhibits retroviral infection soon after the virus enters its target cell by recognizing the capsid of incoming virions through its C-terminal PRYSPRY domain. Different TRIM5α alleles block distinct retroviruses in a species-specific manner: Human TRIM5α (huTRIM5α) potently blocks the so-called N-tropic murine leukemia virus (N-MLV), but is ineffective against the closely related B-tropic (B-MLV) and Moloney (Mo-MLV) strains, or against the simian immunodeficiency virus from macaque (SIVmac) and the two HIV strains (HIV1 and HIV2). In contrast, rhesus macaque TRIM5α (rhTRIM5α) potently blocks HIV1 and HIV2 but not N-, B-, Mo-MLV or SIVmac. My thesis aimed at gaining insight into the molecular mechanism of TRIM5α-mediated retroviral restriction. Firstly by investigating the huTRIM5α specific capture of retroviral capsids to better understand the ability of HIV1 to escape this recognition, and secondly by identifying cellular factors that interact with TRIM5α to subsequently determine their putative role in TRIM5α-mediated antiretroviral activity. In the first set of experiments, I demonstrated that substituting a single amino acid in the PRYSPRY domain of huTRIM5α expands its antiviral activity to B-MLV, Mo-MLV, SIVmac and HIV2, while HIV1 is still able to escape this block. Furthermore, I found that MLV could escape this restriction through the negative influence of four critical residues within its capsid, supporting a model whereby TRIM5α directly interacts with retroviral capsids. In the second part of my thesis, I exploited the remarkable conservation of the capsid among retroviruses to map the four key positions found in MLV onto the HIV1 capsid. I then confirmed that amino-acid substitutions at some of these positions could increase the sensitivity of HIV1 to huTRIM5α restriction. Further mutating an exposed cyclophilin A-binding loop of the HIV1 capsid could reinforce susceptibility to this factor. These results indicate that, despite major sequence divergences, HIV and MLV use structurally homologous residues to avoid capture by huTRIM5α. I then investigated the influence of the coiled-coil domain of huTRIM5α on the range of retroviruses recognised by this antiviral factor. I found that the PRYSPRY domain is necessary but not sufficient to dictate TRIM5α specificity as single amino acid changes within the coiled-coil region could modulate the range of targeted retroviruses. We favour a model whereby the coiled-coil domain exerts this effect indirectly, by influencing the orientation of the PRYSPRY domain and hence its ability to recognize retroviral capsids. Finally, I screened a human cDNA library for cellular proteins that interact with huTRIM5α. I found several hits, the physiological roles of which impact on cellular processes as diverse as ubiquitination and endocytosis. These results open possible avenues to pursue in future studies aimed at deciphering further the mechanisms by which TRIM5α blocks retroviruses.

EPFL2010

Innate Barriers to Genome Invasion

Pierre Maillard

EPFL2010