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Abstract: Perturbation of the human copper-transporter Wilson disease protein (ATP7B) causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the nucleotide-binding subdomain (N-domain), including the most common mutation, H1069Q. To gain insight into the biophysical behavior of the N-domain under normal and disease conditions, we have characterized wild-type and H1069Q recombinant N-domains in vitro and in silico. The mutant has only twofold lower ATP affinity compared to that of the wild-type N-domain. Both proteins unfold in an apparent two-state reaction at 20 °C and ATP stabilizes the folded state. The thermal unfolding reactions are irreversible and, for the same scan rate, the wild-type protein is more resistant to perturbation than the mutant. For both proteins, ATP increases the activation barrier towards thermal denaturation. Molecular dynamics simulations identify specific differences in both ATP orientation and protein structure that can explain the absence of catalytic activity for the mutant N-domain. Taken together, our results provide biophysical characteristics that may be general to N-domains in other P1B-ATPases as well as identify changes that may be responsible for the H1069Q WD phenotype in vivo. [Copyright &y& Elsevier]

Abstract: Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression. [Copyright &y& Elsevier]

[Display omitted] • Compared performance of MM/GBSA and FEP+ in predicting stability of protein variants. • Validated on a dataset of 328 experimentally characterized single mutants.. • FEP+ has R 2 = 0.65 and MUE = 0.95 kcal/mol and outperforms MM/GBSA. • Correct prediction of mutant charge state is essential for reliable FEP+ predictions • Proposed computational workflow: scan with MM/GBSA, and accurately predict with FEP+. This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products. Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods. In contrast to many of the commonly available computational methods for stability prediction, the Free Energy Perturbation approach applied in this paper explicitly accounts for solvent effects and samples conformational dynamics using a rigorous molecular dynamics simulation process. On the entire validation dataset, consisting of 328 single point mutations spread across 14 distinct protein structures, our results show good overall correlation with experiment with an R 2 of 0.65 and a low mean unsigned error of 0.95 kcal/mol. Application of the FEP approach in conjunction with experimental assessment techniques offers opportunities to lower the time and expense of product development and reduce the risk of costly late-stage failures. [ABSTRACT FROM AUTHOR]