Armin Lahm

Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nε2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this “gain of function phenotype” could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.

Three percent of the worlds population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8+ T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.

A model for the hepatitis C virus envelope glycoprotein E2

Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355–366.

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL‐6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution‐phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL‐6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL‐6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL‐6 variants complexed with IL‐6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL‐6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL‐6, two IL‐6R alpha and two gp130 molecules. We propose here a model representing the IL‐6 receptor complex as hexameric, which might be common to other helical cytokines.

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco‐enzyme consisting of 270 amino acid residues which cleaves single‐stranded RNA and DNA into 5′‐mononucleotides. The X‐ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R‐factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N‐acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha‐helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double‐stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

Binding of the Hepatitis C Virus E2 Glycoprotein to CD81 Is Strain Specific and Is Modulated by a Complex Interplay between Hypervariable Regions 1 and 2

ABSTRACT The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter‐and intra‐individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti‐HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients‘ sera and bind serum antibodies that cross‐react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the ‘mimotopes’ with the highest cross‐reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross‐reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation

Interleukin‐6 (IL‐6) drives the sequential assembly of a receptor complex formed by the IL‐6 receptor (IL‐6R alpha) and the signal transducing subunit, gp130. A model of human IL‐6 (hIL‐6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL‐6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL‐6 and gp130 and therefore essential for IL‐6 signal transduction, were subjected to site‐directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL‐6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild‐type affinity towards hIL‐6‐R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild‐type IL‐6.

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin‐6 (IL‐6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post‐menopausal osteoporosis. IL‐6, a four‐helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low‐affinity IL‐6 receptor (IL‐6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL‐6R alpha. These mutants behave as full and selective IL‐6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL‐6 super‐antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL‐6R alpha. The IL‐6 super‐antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild‐type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL‐6 suggests that it could be possible to design super‐antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Evolution of Bacterial and Archaeal Multicomponent Monooxygenases

We report the results of a comparative analysis of the sequences of multicomponent monooxygenases, a family of enzymes of great interest for bioremediation of contaminated soil. We show that their function, in terms of substrate specificity, can be deduced from their subunit organization and composition, that rearrangements of subunits as well as recruitments of new ones can be used to explain their different properties and functionalities, and that the observed pattern can be rationalized invoking a number of evolutionary events, including horizontal gene transfer. Our analysis highlights the plasticity and modularity of this family of enzymes, which might very well be the reason underlying the extremely rapid emergence of new bacterial strains able to grow on contaminated soils.