Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Classification of Beta-Lactamases and Penicillin Binding Proteins Using Ligand-Centric Network Models

β-lactamase mediated antibiotic resistance is an important health issue and the discovery of new β-lactam type antibiotics or β-lactamase inhibitors is an area of intense research. Today, there are about a thousand β-lactamases due to the evolutionary pressure exerted by these ligands. While β-lactamases hydrolyse the β-lactam ring of antibiotics, rendering them ineffective, Penicillin-Binding Proteins (PBPs), which share high structural similarity with β-lactamases, also confer antibiotic resistance to their host organism by acquiring mutations that allow them to continue their participation in cell wall biosynthesis. In this paper, we propose a novel approach to include ligand sharing information for classifying and clustering β-lactamases and PBPs in an effort to elucidate the ligand induced evolution of these β-lactam binding proteins. We first present a detailed summary of the β-lactamase and PBP families in the Protein Data Bank, as well as the compounds they bind to. Then, we build two different types of networks in which the proteins are represented as nodes, and two proteins are connected by an edge with a weight that depends on the number of shared identical or similar ligands. These models are analyzed under three different edge weight settings, namely unweighted, weighted, and normalized weighted. A detailed comparison of these six networks showed that the use of ligand sharing information to cluster proteins resulted in modules comprising proteins with not only sequence similarity but also functional similarity. Consideration of ligand similarity highlighted some interactions that were not detected in the identical ligand network. Analysing the β-lactamases and PBPs using ligand-centric network models enabled the identification of novel relationships, suggesting that these models can be used to examine other protein families to obtain information on their ligand induced evolutionary paths.     

Trace Metal Levels in Rainbow Trout (Oncorhynchus mykiss) Cultured in Net Cages in a Reservoir and Evaluation of Human Health Risks from Consumption

Although fish consumption has positive health effects, metals accumulated in fish can cause human health risks. In this study, the levels of ten metals in rainbow trout (Oncorhynchus mykiss) farmed in the Keban Dam Reservoir, which has the biggest rainbow trout production capacity in Turkey, were determined and compared with the maximum permissible levels (MPLs). Also, human health risks associated with rainbow trout consumption were assessed. The metal concentrations in rainbow trout were found below the MPLs. The estimated daily intake of each metal was much lower than the respective tolerable daily intake. The target hazard quotient (THQ) for individual metal and total THQ for combined metals did not exceed 1, indicating no health risk for consumers. The cancer risk (CR) value for inorganic arsenic was within the acceptable lifetime risk range of 10⁻⁶ and 10⁻⁴. For carcinogenic and non-carcinogenic effects, the maximum allowable fish consumption rates were high enough to ensure the human health. According to these results, the consumption of rainbow trout farmed in the Keban Dam Reservoir does not pose a risk on human health.

     

Alpha7 helix plays an important role in the conformational stability of PTP1B

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix α7), which forms an allosteric binding site 20 ? away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Brain stem cells as the cell of origin in glioma

Glioma incidence rates in the United States are near 20000 new cases per year, with a median survival time of 14.6 mo for high-grade gliomas due to limited therapeutic options. The origins of these tumors and their many subtypes remain a matter of investigation. Evidence from mouse models of glioma and human clinical data have provided clues about the cell types and initiating oncogenic mutations that drive gliomagenesis, a topic we review here. There has been mixed evidence as to whether or not the cells of origin are neural stem cells, progenitor cells or differentiated progeny. Many of the existing murine models target cell populations defined by lineage-specific promoters or employ lineagetracing methods to track the potential cells of origin. Our ability to target specific cell populations will likely increase concurrently with the knowledge gleaned from an understanding of neurogenesis in the adult brain. The cell of origin is one variable in tumorigenesis, as oncogenes or tumor suppressor genes may differentially transform the neuroglial cell types. Knowledge of key driver mutations and susceptible cell types will allow us to understand cancer biology from a developmental standpoint and enable early interventional strategies and biomarker discovery.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

The Color of My Skin: A Measure to Assess Children's Perceptions of Their Skin Color

The Color of My Skin is an instrument developed to assess children's internalized idea (abstraction) of the color of their skin; their satisfaction with that color; the desire, if any, to change the color of their skin; and their affect regarding their skin color. The assessment is part of a questionnaire utilized in a 3-year longitudinal study that examines psychosocial development, physical health, and behavioral adjustment of Puerto Rican children (N = 257) reared in the Greater Boston area. The results demonstrate that children's internalized representation of their skin color is a construct that can be reliably and validly measured. The children's ratings of their skin color were not associated with their sex, school grade, ethnic identity, the child's or the parent's nativity, or the racial or ethnic compositions of 3 social contexts: their neighborhood, their classmates, and their closest friends. Puerto Rican children did not show a preference for light-colored skin. Moreover, there were no significant differences in self-esteem based on the child's self-reported skin color. The lack of association between self-esteem and skin color was interpreted in light of a developmental tendency prevalent in early to middle childhood to place a positive value on different aspects of one's self. Whereas almost all children (96%) reported being happy or very happy with their color, 16% of the children would like to change their skin color if they could (51% to a lighter and 46% to a darker color).     

Live Cell Imaging of Peptide Uptake Using a Microfluidic Platform

International Journal of Peptide Research and Therapeutics - Cell penetrating peptides (CPPs) are unique molecules with the ability to pass through biological membranes as they carry their cargoes...