Emilia Pedone

Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

Crystal structure of the resuscitation-promoting factor (DeltaDUF)RpfB from M. tuberculosis.

Mycobacterium tuberculosis is able to establish a non-replicating state and survive in an intracellular habitat for years. Resuscitation of dormant M. tuberculosis bacteria is promoted by resuscitation-promoting factors (Rpfs), which are secreted from slowly replicating bacteria close to dormant bacteria. Here we report the crystal structure of a truncated form of RpfB (residues 194-362), the sole indispensable Rpf of the five Rpfs encoded in this bacterium genome. The structure, denoted as (DeltaDUF)RpfB, exhibits a comma-like shape formed by a lysozyme-like globular catalytic domain and an elongated G5 domain, which is widespread among cell surface binding proteins. The G5 domain, whose structure was previously uncharacterised, presents some peculiar features. The basic structural motif of this domain, which represents the tail of the comma-like structure, is a novel super-secondary-structure element, made of two beta-sheets interconnected by a pseudo-triple helix. This intricate organisation leads to the exposure of several backbone hydrogen-bond donors/acceptors. Mutagenesis analyses and solution studies indicate that this protein construct as well as the full-length form are elongated monomeric proteins. Although (DeltaDUF)RpfB does not self-associate, the exposure of structural elements (backbone H-bond donors/acceptors and hydrophobic side chains) that are usually buried in globular proteins is typically associated with adhesive properties. This suggests that the RpfB G5 domain has a cell-wall adhesive function, which allows the catalytic domain to be properly oriented for the cleavage reaction. Interestingly, sequence comparisons indicate that these structural features are also shared by G5 domains involved in biofilm formation.

Structure and Functional Regulation of RipA, a Mycobacterial Enzyme Essential for Daughter Cell Separation

Cell separation depends on cell-wall hydrolases that cleave the peptidoglycan layer connecting daughter cells. In Mycobacterium tuberculosis, this process is governed by the predicted endopeptidase RipA. In the absence of this enzyme, the bacterium is unable to divide and exhibits an abnormal phenotype. We here report the crystal structure of a relevant portion of RipA, containing its catalytic-domain and an extra-domain of hitherto unknown function. The structure clearly demonstrates that RipA is produced as a zymogen, which needs to be activated to achieve cell-division. Bacterial cell-wall degradation assays and proteolysis experiments strongly suggest that activation occurs via proteolytic processing of a fully solvent exposed loop identified in the crystal structure. Indeed, proteolytic cleavage at this loop produces an activated form, consisting of the sole catalytic domain. Our work provides the first evidence of self-inhibition in cell-disconnecting enzymes and opens a field for the design of novel antitubercular therapeutics.

Molecular organization of the cullin E3 ligase adaptor KCTD11

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (KM 0.3 µm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.

Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress

Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome database of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Its role in oxidative stress was investigated by transcriptional analysis of RNA isolated from cultures that had been stressed with various oxidant agents. Its specific involvement was confirmed by a considerable increase in the bcp2 transcript following induction with H2O2. The 5′ end of the transcript was mapped by primer extension analysis and the promoter region was characterized. bcp2 was cloned and expressed in Escherichia coli, the recombinant enzyme was purified and the predicted molecular mass was confirmed. Using dithiothreitol as an electron donor, this enzyme acts as a catalyst in H2O2 reduction and protects plasmid DNA from nicking by the metal‐catalysed oxidation system. Western blot analysis revealed that the Bpc2 expression was induced as a cellular adaptation in response to the addition of exogenous stressors. The results obtained indicate that Bcp2 plays an important role in the peroxide‐scavaging system in S. solfataricus. Mutagenesis studies have shown that the only cysteine, Cys49, present in the Bcp2 sequence, is involved in the catalysis. Lastly, the presence of this Cys in the sequence confirms that Bcp2 is the first archaeal 1‐Cysteine peroxiredoxin (1‐Cys Prx) so far identified.

The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain.

The first putative prokaryotic Cys2His2 zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys2His2 zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys2His2 zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9–66), is arranged in a βββαα topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain.

Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in thermal denaturation pathway of the proteins: an infrared spectroscopic study.

The structure of thioredoxin from Alicyclobacillus acidocaldarius (previously named Bacillus acidocaldarius ) (BacTrx) and from Escherichia coli ( E. coli Trx) was studied by Fourier-transform IR spectroscopy. Two mutants of BacTrx [Lys(18)-->Gly (K18G) and Arg(82)-->Glu (R82E)] were also analysed. The data revealed similar secondary structures in all proteins, but BacTrx and its mutants showed a more compact structure than E. coli Trx. In BacTrx and its mutants, the compactness was p(2)H-dependent. All proteins revealed the existence of a molten globule-like state. At p(2)H 5.8, the temperature at which this state was detected was higher in BacTrx and decreased in the different proteins in the following order: BacTrx>R82E>K18G> E. coli Trx. At neutral or basic p(2)H, the molten globule-like state was detected at the same temperature in both BacTrx and R82E, whereas it was found at the same temperature in all p(2)Hs tested for E. coli Trx. The thermal stability of the proteins was in the following order at all p(2)Hs tested: BacTrx>R82E>K18G> E. coli Trx, and was lower for each protein at p(2)H 8.4 than at neutral or acidic p(2)Hs. The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p(2)Hs tested, whereas they were present in R82E and E. coli Trx samples only at p(2)H 5.8. The results indicated that a single mutation might affect the structural properties of a protein, including its propensity to aggregate at high temperatures. The data also indicated a possible application of Fourier-transform IR spectroscopy for assessing molten globule-like states in small proteins.

SENSING AND ADAPTING TO ENVIRONMENTAL STRESS: THE ARCHAEAL TACTIC

Archaea occupy a considerable diversity of niches encompassing extreme environments with extreme of pH, salinity and temperature that cannot be tolerated by other forms of life. Survival and colonisation requires the capacity to sense, and adapt to environmental change. In this review we consider the issues of adaptation to environmental stresses, in particular the mechanisms that might be employed by different Archaea to respond to the specific challenges of their particular niche. We lay emphasis on the strategies adopted to respond to oxidative and chemical stress. In particular, this paper reviews major key points in the generation of reactive oxygen species in Archaea, defense mechanisms and genetic responses to oxidative stress. Finally, we discuss complex biological response mechanisms to chemical damage with particular attention to detoxification from metals and drugs.

Peroxiredoxins as cellular guardians in Sulfolobus solfataricus: characterization of Bcp1, Bcp3 and Bcp4.

Peroxiredoxins are ubiquitous enzymes that are part of the oxidative stress defense system. In the present study, we identified three peroxiredoxins [bacterioferritin comigratory protein (Bcp)1, Bcp3 and Bcp4] in the genome of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Based on the cysteine residues conserved in the deduced aminoacidic sequence, Bcp1 and Bcp4 can be classified as 2‐Cys peroxiredoxins and Bcp3 as a 1‐Cys peroxiredoxin. A comparative study of the recombinant Bcps produced in Escherichia coli showed that these enzymes protect DNA plasmid from oxidative damage and remove both H2O2 and tert‐butyl hydroperoxide, although at different efficiencies. We observed that all of them were particularly thermostable and that peak enzymatic activity fell within the range of the growth temperature of S. solfataricus. Furthermore, we discovered an alternative Bcp reduction system whose composition differs from that of the peroxiredoxin reduction system previously characterized in the aerobic hyperthermophilic archaeon Aeropyrum pernix. Whereas the latter uses the thioredoxin/thioredoxin reductase/NADPH system, this alternative Bcp system is formed of the protein disulfide oxidoreducatase, SSO0192, the thioredoxin reductase, SSO2416, and NADPH. The role of Bcps in oxidative stress was investigated using transcriptional analysis. Different northern blot analysis responses suggested that the Bcp antioxidant system of S. solfataricus can both operate at the constitutive level, with Bcp1 and Bcp4 preventing endogenous peroxide formation, and at the inducible level, with Bcp3 and the already characterized Bcp2 protecting cells from the attack of external peroxides.