Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Elevational patterns of carbon,nitrogen and phosphorus in understory bryophytes on the eastern slope of Gongga Mountain,China

Aims The nutrient uptake, requirement and releasing rates of bryophytes are very different from those of tracheophytes. However, it is difficult to make a quantitative evaluation of bryophytes’ roles in nutrient cycling and their specific eco-physiological adaptations due to lack of knowledge of their concentrations and stoichiometric ratios of carbon (C), nitrogen (N) and phosphorus (P). To fill this gap, the present study aims to investigate: (i) what are the elevational trends of C, N and P concentrations and stoichiometric ratios of bryophytes? (ii) whether C, N and P concentrations and stoichiometric ratios of bryophytes differ between different bryophyte types (in terms of the growth form and living substrate)? and (iii) how do the exponent scalings of N and P of bryophytes change along the elevational gradient?     

Carbon: terrestrial C4 plants

The carbon isotope composition of terrestrial C₄ plants depends on the primary carboxylation of phosphoenolpyruvate (PEP) and on the diffusion of CO₂ to the carboxylation sites, but is also influenced by the final carboxylation of ribulose-1,5-bisphosphate (RuBP). Several models have been used for reproducing this complex situation. In the present review, a particular model is applied as a means to interpret the effects of environmental and genetically determined factors on carbon isotope discrimination during C₄ photosynthesis. As a new feature, the model considers four types of limitation of the overall CO₂ assimilation rate. Both carboxylation reactions are assumed to be limited by either maximum enzyme activity or maximum substrate regeneration rate. The model is applied to experimental data on the effects of CO₂, irradiance and water stress on short-term discrimination by leaves of several C₄ species measured simultaneously with CO₂ gas exchange characteristics. In particular, different patterns of the influence of low irradiances on carbon isotope discrimination are interpreted as due to variations in that irradiance at which a transition from limitation by PEP regeneration rate and RuBP carboxylase activity to limitation by the regeneration rates of both substrates occurs. After discussing literature data on the effects of environmental conditions on carbon isotope discrimination by C₄ plants seasonal and developmental changes in carbon isotope composition, studies on the systematic and geographic distribution of C₄ plants, evolutionary and genetical aspects, and some ecological implications are reviewed.     

Substrates for clinical applicability of stem cells

The capability of human pluripotent stem cells(h PSCs) to differentiate into a variety of cells in the human body holds great promise for regenerative medicine. Many substrates exist on which h PSCs can be self-renewed, maintained and expanded to further the goal of clinical application of stem cells. In this review, we highlight numerous extracellular matrix proteins, peptide and polymer based substrates, scaffolds and hydrogels that have been pioneered. We discuss their benefits and shortcomings and offer future directions as well as emphasize commercially available synthetic peptidesas a type of substrate that can bring the benefits of regenerative medicine to clinical settings.     

Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases

S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington''s disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

Substrate moisture,particle size and temperature preferences of trap‐building larvae of sympatric antlions and wormlions from the rainforest of Borneo

1. Larvae of antlions (Neuroptera: Myrmeleontidae) and wormlions (Diptera: Vermileonidae) display a convergently evolved sit‐and‐wait hunting strategy of building pitfall traps in sandy areas. This study investigated a sympatric population of antlions and wormlions in the lowland rainforest of Borneo for substrate moisture, particle size and temperature preferences. It was hypothesised that these animals would show different preferences regarding these microhabitat traits. 2. The results showed that antlions had a higher aversion to moisture compared with wormlions, but that wormlions had a higher preference for small‐particle sand. Furthermore, thermal preferences in antlions and wormlions were significantly different, with antlions choosing higher temperatures. 3. The detected differences between antlions and wormlions might contribute to their niche partitioning in the mixed Bornean population and thus facilitate coexistence of these animals. It is possible that the hotter and dryer microhabitat edges are preferred by antlions.     

Influence of soil moisture on supercooling capacity and associated physiological parameters of overwintering larvae of rice stem borer

Influence of soil moisture on the supercooling capacity and associated physiological parameters of overwintering larvae of the rice stem borer Chilo suppressalis was examined by exposing larvae to soil moistures of 25, 50, 75 and 100% of saturated soil water content (SSWC) at ambient temperature for 30 d from December 2007 to January 2008 in Beijing, China. At the end of the exposure, supercooling points (SCPs) varied significantly among the treatments, the lowest being in the larvae exposed to soil moisture of 25% SSWC. Fresh weight was significantly higher in the larvae exposed to soil moisture of 100% SSWC than in those kept at 25 and 50% SSWC. Dry weight and body water content (% fresh weight) were not different among the treatments. Glucose and trehalose contents were markedly lower, and glycerol content was significantly higher in the larvae confined to soil moisture of 25% SSWC than in those exposed to the other soil moisture treatments. It is suggested that variation in body water content (% fresh weight) contributes to the differences in SCPs of the overwintering C. suppressalis larvae in all treatments, but the influence of soil moisture treatments on supercooling capacity are caused through changes in glycerol content.     

Effects of Dinner Composition on Postprandial Macronutrient Oxidation in Prepubertal Girls

Objective: To see whether a fat‐rich (50%) evening meal promoted fat oxidation and a different spontaneous food intake on the following day at breakfast than a meal with a lower fat content (20%) in 10 prepubertal obese girls. Research Methods and Procedures: The postabsorptive and postprandial (10.5 hours) energy expenditure after a low‐fat (LF) (20% fat, 68% carbohydrate, 12% protein) and an isocaloric (2.1 MJ) and isoproteic high‐fat (HF; 50% fat, 38% carbohydrate, 12% protein) meal were measured by in direct calorimetry. Results: Fat oxidation was not significantly different after the two meals [LF, 31 ± 9 vs. HF, 35 ± 9 g/10.5 hours, p = not significant (NS)]. The girls oxidized 1.8 ± 0.9 times more fat than that ingested (11.1 grams) with the LF meal vs. 0.3 ± 0.3 times more fat than that ingested (27.1 grams) with the HF meal (p < 0.001). Carbohydrate oxidation was significantly higher after an LF than an HF meal (39 ± 12 vs. 29 ± 9 g/10.5 hours, p < 0, 05). At breakfast, the girls spontaneously ingested a similar amount of energy (1.5 ± 0.7 vs. 1.5 ± 0.6 MJ, p = NS) and macronutrient proportions (fat, 23% vs. 26%, p = NS; protein, 9% vs. 10%; carbohydrate, 68% vs. 64%,) independently of their having eaten an HF or an LF dinner. Discussion: An HF dinner did not stimulate fat oxidation, and no compensatory effect in spontaneous food intake was observed during breakfast the following morning. Cumulated total fat oxidation after dinner was higher than total fat ingested at dinner, but a much larger negative fat balance was observed after the LF meal. Spontaneous energy and nutrient intakes at breakfast were similar after LF and HF isocaloric, isoproteic dinners. This study points out the lack of sensitivity of short‐term fat balance to subsequently readjust fat intake and emphasizes the importance of an LF meal to avoid transient positive fat imbalance.