Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.     

Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser⁹⁹¹ and Tyr⁹⁹⁸ accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser¹⁰³⁹ and Thr¹⁰⁴¹ were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr⁹⁹⁸, Ser⁹⁹¹, Ser¹⁰³⁹, and Thr¹⁰⁴¹ governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.Upon activation by ligand, the epidermal growth factor receptor (EGFR)¹ dimerizes, sometimes as heterodimers with other EGFR family members; is catalytically activated by reorientation of kinase region subdomains; becomes covalently modified by phosphorylation and ubiquitination; and interacts with a variety of intracellular proteins (1, 2). These events activate intracellular signaling cascades, and concurrently the dimerized receptors become internalized through endocytosis and then may be recycled to the cell surface or degraded in lysosomes (3). Systematic analysis of EGFR family phosphorylation-dependent protein interactions has been assessed (4, 5), and many of the known EGFR-interacting proteins can be categorized as functioning in cellular processes such as EGF-induced signal transduction and EGFR endocytosis and trafficking. Temporal analysis of tyrosine phosphorylation following EGF treatment of cells revealed groups of EGFR substrates with shared profiles of phosphorylation kinetics, including some that display rapid kinetics of phosphorylation accumulation and are involved in signal transduction (e.g. ERK kinase activation) and others that accumulate more slowly following ligand treatment and are involved in receptor internalization and down-regulation (5–11). Although advances in MS and the definition of phosphorylation-dependent protein-protein interactions have led to a greatly expanded view of EGFR function and regulation, our understanding of the biological consequences and spatial-temporal relationships of individual modifications is incomplete.In a previous quantitative phosphoproteomics study aimed at the identification of drug-modulated changes in phosphorylation associated with the EGFR network, a cluster of three sites of phosphorylation in the EGFR carboxyl tail region was identified as affected by receptor stimulation by EGF and inhibited by the ATP-competitive EGFR inhibitor PKI166 in human A431 tumor cells and xenograft tumors (12). The three sites in the cluster, Ser⁹⁹¹,² Ser⁹⁹⁵, and Tyr⁹⁹⁸, are localized within a single tryptic peptide having the sequence MHLPSPTDSNFYR that spans residues 987–999. The phosphorylation of Tyr⁹⁹⁸ was first described by Stover et al. (12), whereas the two serine sites were shown previously to be phosphorylated by Heisermann and Gill (13). Numerous recent studies using different cultured cell models have verified the phosphorylation of EGFR at Tyr⁹⁹⁸ and Ser⁹⁹¹ (10, 11, 14, 15), and Thr⁹⁹³ was also observed to be phosphorylated within this same region of the EGFR in EGF-stimulated HeLa cells (10). The modulation of these sites by EGF and the EGFR inhibitor implicates them in EGFR signaling and suggests that they may have utility as pharmacodynamic markers of EGFR activity. However, the function and importance of these sites, their modulation by kinases and phosphatases, and possible roles in EGFR function remain unknown.Several amino acid residues in the EGFR have been implicated in the regulation of its trafficking. Sorkin et al. (16) showed that substitution of Tyr⁹⁹⁸ with phenylalanine rendered high density EGFRs defective for endocytosis and interaction with AP-2. More recent kinetic studies using MS indicated that EGFR phosphorylation at both Tyr⁹⁹⁸ (5) and Ser⁹⁹¹ (10) occurs relatively slowly compared with other EGF-induced tyrosine phosphorylations known to be involved in receptor-proximal signal transduction. For example, Mann and co-workers (10) recorded maximal phosphorylation at EGFR sites Tyr¹¹¹⁰, Tyr¹¹⁷², and Tyr¹¹⁹⁷ at 1 min post-EGF, whereas EGF-stimulated phosphorylation at Tyr⁹⁹⁸ was still increasing at 15 min post-EGF (5), and a peptide containing both Ser(P)⁹⁹¹ and Thr(P)⁹⁹³ peaked after 10 min (10). However, the role of phosphorylation at Tyr⁹⁹⁸ and Ser⁹⁹¹ has not been reported. Another region of the EGFR, spanning residues 1026–1046, was identified by Zwang and Yarden (17) as a target of phosphorylation downstream of the stress-activated mitogen-activated protein (MAP) kinase p38 and associated with transient internalization and recycling of the EGFR in response to cytokine (TNFα) and stress challenges such as UV irradiation and the chemotherapeutic agent cisplatinum. Within this part of the receptor, a 13-residue section spanning 1029–1041 and the leucines at 1034 and 1035 in particular were found to be essential for ligand- and dimerization-induced EGFR endocytosis (18). Although both EGF- and stress-induced EGFR internalization may be clathrin-mediated, they differ in that the former leads to receptor down-regulation and involves the E3 ubiquitin ligase CBL (19), whereas the latter involves receptor recycling, is not associated with receptor phosphorylation at the CBL binding site Tyr(P)¹⁰⁶⁹, and, in the case of TNFα treatment, involves activation of the transforming growth factor β-activated kinase TAK1 upstream of p38 (20). Interestingly although p38 kinase is not required for EGF-induced EGFR internalization, it is required for CBL-dependent receptor degradation (21). Therefore, alternate pathways involving p38 kinase regulate the down-regulation or recycling of the EGFR in response to diverse extracellular signals. However, the molecular details that govern these two processes are not fully understood.In the current study, EGFR phosphorylation, signaling, protein-protein interactions, and trafficking were analyzed to address the role of Tyr⁹⁹⁸ and Ser⁹⁹¹ in EGFR endocytosis. This was achieved by application of complementary methods including quantitative selected reaction monitoring (SRM, also known as MRM for multiple reaction monitoring) mass spectrometry, fluorescence imaging and cell sorting, immunoaffinity protein enrichment and blotting, and site-directed mutagenesis. Substitution mutations that prevented phosphorylation at EGFR Tyr⁹⁹⁸ and Ser⁹⁹¹ did not prevent EGFR-to-ERK signaling but impaired EGF-induced receptor internalization and stimulated p38 kinase-dependent receptor phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These findings confirm the importance of Tyr⁹⁹⁸ and reveal a role for Ser⁹⁹¹ in EGF-mediated EGFR internalization possibly involving cross-talk with the p38 kinase-dependent EGFR recycling pathway.     

Fibronectin stimulates TRPV1 translocation in primary sensory neurons

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin (FN) plays in the dynamic modulation of transient receptor potential family V type 1 receptor (TRPV1) translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind FN. TG neurons cultured upon or treated with FN experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This FN-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on FN demonstrated an increase in capsaicin-mediated Ca²⁺ accumulation relative to neurons cultured on poly- d -lysine. Data presented from these studies indicate that FN stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying FN as a critical component of the ECM capable of sensory neuron sensitization.     

Growth performance of Ganoderma lucidum using billet method in Garhwal Himalaya,India

Medicinal mushrooms have been used in various treatments from a very long time, among which, Ganoderma lucidum is one of the most important medicinal mushroom. It is cultivated worldwide to meet its ever-increasing demand in the market. It is generally cultivated by bed log (Sawdust) and wood log (billet) method. This study was an attempt to observe the growth performance of G. lucidum on poplar billets (Populus deltoides) in the Sherpur Village (Dehradun) and Manjgaun village (Tehri Garhwal) of Garhwal Himalaya, India. The farmers’ field with empty house/ rooms having proper growing conditions especially humidity and light were used for the cultivation of G. lucidum. The G. lucidum spawn was inoculated in poplar wood billets and these billets were installed in well prepared soil. The results demonstrated that cropping cycle of G. lucidum was shorter (132–136 days) in Sherpur Village (Dehradun) as compared to Manjgaun village (141–145 days) in Tehri Garhwal. Further the results also revealed that yield was decreased in the subsequent flushes. In Village Sherpur, the fruiting bodies of G. lucidum were harvested between 64-66 days, 100-101  days and 135-136  days during first, second and third flush after the installation of billets, respectively. However; in village Manjgaun, the fruiting bodies of G. lucidum were harvested between 69 and 71 days, 107-108  days and 144-145 days in first, second and third after the installation of billets respectively. Warmer temperature in Village Sherpur resulted in the early emergence and development of the fruiting bodies as compared to village Manjgaun where pinhead and fruiting body development was delayed due to the lower temperature during cropping cycle.