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.

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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.

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.

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.

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR ⌬43 ) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR ⌬43 (L6 IR⌬43 ) 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 kinasedependent coprecipitation of PDK-1 with wild-type IR (IR WT ), but not IR ⌬43 . Based on overlay blotting, PDK-1 directly bound IR WT , but not IR ⌬43 . Insulin-activated IR WT , and not IR ⌬43 , 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. Tyr3Phe 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 IR⌬43 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.The interaction of insulin with its cell surface receptors leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase, or PI 3-K) and generation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] at the inner surface of the cell membrane (19). PtdIns(3,4,5)P 3 generation leads to the activation of a group of AGC family protein kinases, including different protein kinase B (PKB)/Akt isoforms, p70 ribosomal S6 kinase, serum-and glucocorticoid-induced protein kinase, and atypical protein kinase C (1,9,11,12,24,28,33). These kinases play key roles in the regulation of cell metabolism, proliferation, and survival by insulin, as well as other growth factors (4). How the AGC kinases are activated following insulin stimulation of PI 3-kinase has been extensively investigated (41). PKB/Akt activation requires phosphorylation at two highly conserved Ser/Thr residues by 3-phosphoinositidedependent protein kinase 1 (PDK-1) (22) and by a distinct 3-phosphoinositide-dependent protein kinase 2 (PDK-2), recently identified as the mammalian target of rapamycin (21, 10, 36). Thus, PDKs represent master regulators of AGC kinase signal transduction (38,40,44). PDK-1 is composed of a Cterminal PH domain and a catalytic domain similar to the catalytic domains of PKA, PKB, and PKC (3). After stimulation by insulin, PI 3-kinase-generated lipids bind the PH domain of PDK-1, promoting its translocation to the membrane, together with that of other PH dom...

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.

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.