Survival of hypoxia-induced dormancy is not a common feature of all strains of the Mycobacterium tuberculosis complex.

While persistence in a dormant state is crucial for the life cycle of Mycobacterium tuberculosis, no investigation regarding dormancy survival of different strains across different lineages was performed so far. We analyzed responses to oxygen starvation and recovery in terms of growth, metabolism, and transcription. All different strains belonging to the Euro-American lineage (L4) showed similar survival and resuscitation characteristics. Different clinical isolates from the Beijing (L2), East African-Indian (L3), and Delhi/Central Asian (L1) lineage did not survive oxygen starvation. We show that dormancy survival is lineage-dependent. Recovery from O2 starvation was only observed in strains belonging to the Euro-American (L4) lineage but not in strains belonging to different lineages (L1, L2, L3). Thus, resuscitation from dormancy after oxygen starvation is not a general feature of all M. tuberculosis strains as thought before. Our findings are of key importance to understand infection dynamics of non-Euro-American vs Euro-American strains and to develop drugs targeting the dormant state.

Cell division site placement and asymmetric growth in mycobacteria.

Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.

Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-kappaB activation.

The molecular complex containing BCL10 and CARMA [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase)] proteins has recently been identified as a key component in the signal transduction pathways that regulate activation of the transcription factor NF-kappaB (nuclear factor kappaB) in lymphoid and non-lymphoid cells. Assembly of complexes containing BCL10 and CARMA proteins relies on homophilic interactions established between the CARDs of these proteins. In order to identify BCL10-inhibitory peptides, we have established a method of assaying peptides derived from the CARD of BCL10 in binding competition assays of CARD-CARD self-association. By this procedure, a short peptide corresponding to amino acid residues 91-98 of BCL10 has been selected as an effective inhibitor of protein self-association. When tested in cell assays for its capacity to block NF-kappaB activation, this peptide represses activation of NF-kappaB mediated by BCL10, CARMA3 and PMA/ionomycin stimulation. Collectively, these results indicate that residues 91-98 of BCL10 are involved in BCL10 self-association and also participate in the interaction with external partners. We also show that blocking of the CARD of BCL10 may potentially be used for the treatment of pathological conditions associated with inappropriate NF-kappaB activation.

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.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a resuscitation-promoting factor from Mycobacterium tuberculosis.

The resuscitation-promoting factor RpfB, the most complex of the five resuscitation-promoting factors produced by M. tuberculosis, is devoted to bacterial reactivation from the dormant state. RpfB consists of 362 residues predicted to form five domains. An RpfB fragment containing the protein catalytic domain and a G5 domain has been successfully crystallized using vapour-diffusion methods. This is the first crystallographic study of a resuscitation-promoting factor. Crystals of this protein belong to space group I422, with unit-cell parameters a = 97.63, b = 97.63, c = 114.87 A. Diffraction data have also been collected from a selenomethionine derivative at 2.9 A resolution. Model building using the phases derived from the multiwavelength anomalous dispersion experiment is in progress.

Does tetracycline bind helix 2 of prion? An integrated spectroscopical and computational study of the interaction between the antibiotic and alpha helix 2 human prion protein fragments.

We demonstrate here that tetracycline (TC) can strongly interact (KD' = 189 +/- 7 nM) with model peptides derived from the C-terminal globular domain of the prion protein, hPrP [173-195], and that interaction concerns residues within the C-terminal half of the helix 2, a short region previously indicated as endowed with ambivalent conformational behavior and implicated in PrP conversion to the beta-sheet-rich, infective scrapie variant. Data have been confirmed by binding studies with the N-terminal truncated 180-195 variant that displays a dissociation constant of 483 +/- 30 nM. Remarkably, TC does not influence the structure of the N-terminally fluoresceinated peptides that both show alpha-helical conformations. Docking calculations and molecular dynamics simulations suggest a direct, strong interaction of the antibiotic with exposed side chain functional groups of threonines 190-193 on the solvent-exposed surface of helix 2.

Effect of salts on the structural behavior of hPrP alpha2-helix-derived analogues: the counterion perspective.

Both theoretical studies and direct experimental evidence have emphasized the importance of electrostatic interactions in the general phenomenon of spontaneous amyloid fibril formation. A number of observations have recently spurred interest in the contribution of these interactions to the conformational behavior of the prion protein. In this paper, we show how salt addition and pH change can modify the conformation of two peptide analogues derived from the human prion protein helix 2 according to a Hofmeister-series-type dependence. Employment of various sodium salts allowed us to highlight the fact that chaotropic anions favor unstructured conformation, whereas kosmotropic anions promote the formation of compact structures like alpha-helix and beta-sheet, which may ultimately facilitate fibril formation. This finding should warn people engaged in ion-based research on prion and derived peptides about cation-bound effects, which have been almost exclusively investigated to date, being easily confounded with modifications that are actually caused by anion activity, thus leading researchers into misunderstand ion-specific effects. To avoid the common complication of ion confounding, it is highly desirable that experiments be designed so that the species causing the modification can be unequivocally perceived.

The prion protein: Structural features and related toxic peptides.

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrP(C)) into an insoluble, partially protease-resistant abnormal scrapie form (PrP(Sc)). PrP(C) is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrP(C) to PrP(Sc) parallels a conformational change of the polypeptide from a predominantly alpha-helical to a highly beta-sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure-activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N-terminal and globular C-terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high beta-sheet propensity and tendency to form beta-sheet-like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.

The effect of the osmolyte trimethylamine N-oxide on the stability of the prion protein at low pH.

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.

Structural determinants of unexpected agonist activity in a retro-peptide analogue of the SDF-1alpha N-terminus.

We have synthesised two retro-peptide analogues of the stromal cell derived growth factor 1 (SDF-1alpha) segment known to be critical for CXCR4 receptor binding, corresponding to the sequences HSEFFRCPCRFFESH and HSEFFRGGGRFFESH. We have assayed the ability of these peptides to activate extracellular signal-regulated kinase 1/2 phosphorylation in cells over expressing the SDF-1alpha receptor, finding that the first variant was able to serve as an agonist of CXCR4, whereas the second one was inactive. Finally, by comparing representative solution structures of the two peptides, we have found that the biological response of HSEFFRCPCRFFESH may be ascribed to a beta-beta-type turn motif centred on Phe(4)-Phe(5).

The human prion protein alpha2 helix: a thermodynamic study of its conformational preferences.

We have synthesized both free and terminally-blocked peptide corresponding to the second helical region of the globular domain of normal human prion protein, which has recently gained the attention of structural biologists because of a possible role in the nucleation process and fibrillization of prion protein. The profile of the circular dichroism spectrum of the free peptide was that typical of alpha-helix, but was converted to that of beta-structure in about 16 h. Instead, below 2.1 x 10(-5) M, the spectrum of the blocked peptide exhibited a single band centered at 200 nm, unequivocally associated to random conformations, which did not evolve even after 24 h. Conformational preferences of this last peptide have been investigated as a function of temperature, using trifluoroethanol or low-concentration sodium dodecyl sulfate as alpha- or beta-structure inducers, respectively. Extrapolation of free energy data to zero concentration of structuring agent highlighted that the peptide prefers alpha-helical to beta-type organization, in spite of results from prediction algorithms. However, the free energy difference between the two forms, as obtained by a thermodynamic cycle, is subtle (roughly 5-8 kJ mol(-1) at any temperature from 280 K to 350 K), suggesting conformational ambivalence. This result supports the view that, in the prion protein, the structural behavior of the peptide is governed by the cellular microenvironment.