Modulation of a protein free-energy landscape by circular permutation

Circular permutations usually retain the native structure and function of a protein while inevitably perturb its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, while subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, where one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild-type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed

Identification of a Novel Mcl-1 Protein Binding Motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins

Studio delle concentrazioni di leptina e adiponectina nella gravidanza fisiologica: ruolo nella sindrome metabolica

Obiettivo: valutare le concentrazioni di leptina e adiponectina nel sangue materno e fetale e le eventuali correlazioni con indici metabolici e di crescita. Pazienti e metodi: sono state studiate 20 gestanti con gravidanza fisiologica al III trimestre. Campioni di plasma materno e neonatale sono stati raccolti al momento del parto e a tre giorni dal parto. Risultati: le concentrazioni materne di leptina e adiponectina si riducono significativamente dopo il parto. La concentrazione media di leptina si riduce significativamente a 72 ore dal parto nel sangue neonatale, mentre non si modifica la concentrazione media di adiponectina. È presente una correlazione positiva tra leptina ed adiponectina nel sangue fetale. L’adiponectina fetale si correla con alcuni parametri biometrici (circonferenza del cranio) e biochimici (colesterolo, trigliceridi) neonatali. Conclusioni: la diversa correlazione nel feto rispetto all’adulto può essere legata al diverso metabolismo degli acidi grassi nel periodo prenatale o ai fattori che regolano la secrezione dell’adiponectina a livello dell’adipocita fetale. Il ruolo di queste due adipochine durante la gravidanza e la crescita e lo sviluppo fetali non è noto, tuttavia i dati suggeriscono una loro partecipazione nei sistemi che regolano nel feto l’omeostasi energetica e il controllo del feed-back endocrino tra tessuto adiposo, sistema nervoso centrale e organi/tessuti periferici (pancreas, muscoli scheletrici, ecc.)