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Nuclear localization signal (NLS) sequence from capsid protein of Venezuelan equine encephalitis virus (VEEV) binds to importin- α transport protein and clogs nuclear import. Prevention of viral NLS binding to importin- α may represent a viable therapeutic route. Here, we investigate the molecular mechanism by which two diffusively binding inhibitors, DP9 and DP9o, interfere with the binding of VEEV’s NLS peptide to importin- α . Our study uses all-atom replica exchange molecular dynamics simulations, which probe the competitive binding of the VEEV NLS fragment, the coreNLS peptide, and the inhibitors to importin- α . Our previous simulations of non-competitive binding of the coreNLS, in which it natively binds to importin- α , are used as a reference. Both inhibitors abrogate native peptide binding and reduce the fraction of its native interactions, but they fail to prevent its non-native binding to importin- α . As a result, these inhibitors turn the coreNLS into diffusive binder, which adopts a manifold of non-native binding poses. Competition from the inhibitors compromises the free energy of coreNLS binding to importin- α showing that they reduce its binding affinity. The inhibition mechanism is based on masking the native binding interactions formed by the coreNLS amino acids. Surprisingly, ligand interference with the binding interactions formed by importin- α amino acids contributes little to inhibition. We show that DP9 is a stronger inhibitor than DP9o. By comparative analysis of DP9 and DP9o interactions we determine the atomistic reason for a relative “success” of DP9, which is due to the intercalation of this inhibitor between the side chains of NLS lysine residues. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for the inhibitors. AlphaScreen data confirmed in silico ranking of the inhibitors. By combining our recent studies, we discuss the putative mechanism by which diffusively binding inhibitors impact protein-protein interactions.

The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fast-twitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.