Essential role of Hrs in a recycling mechanism mediating functional resensitization of cell signaling

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is well known to terminate cell signaling by sorting activated receptors to the MVB/lysosomal pathway. Here we identify a distinct role of Hrs in promoting rapid recycling of endocytosed signaling receptors to the plasma membrane. This function of Hrs is specific for receptors that recycle in a sequence-directed manner, in contrast to default recycling by bulk membrane flow, and is distinguishable in several ways from previously identified membrane-trafficking functions of Hrs/Vps27p. In particular, Hrs function in sequence-directed recycling does not require other mammalian Class E gene products involved in MVB/lysosomal sorting, nor is receptor ubiquitination required. Mutational studies suggest that the VHS domain of Hrs plays an important role in sequence-directed recycling. Disrupting Hrs-dependent recycling prevented functional resensitization of the beta(2)-adrenergic receptor, converting the temporal profile of cell signaling by this prototypic G protein-coupled receptor from sustained to transient. These studies identify a novel function of Hrs in a cargo-specific recycling mechanism, which is critical to controlling functional activity of the largest known family of signaling receptors.     

AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling,and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity

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Molecular dynamics simulations of apo,holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

The pyridoxal 5‐phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ‐aminobutyric acid aminotransferase (GABA‐AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA‐AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA‐AT. This is a consequence of a pK_a shift triggered by a strong charge–charge interaction with an ionic “diad” formed by Asp298 and His190 that would help the activation of the first half‐reaction of the catalytic mechanism in GABA‐AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π–π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half‐reaction in GABA‐AT: the regeneration of PLP‐bound GABA‐AT (i.e., the holoenzyme). Proteins 2016; 84:875–891. © 2016 Wiley Periodicals, Inc.     

PURIFICATION AND CHARACTERIZATION OF β‐GLUCOSIDASE FROM GREATER WAX MOTH Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The greater wax moth, Galleria mellonella, is one of the most ruinous pests of honeycomb in the world. Beta‐glucosidases are a type of digestive enzymes that hydrolytically catalyzes the beta‐glycosidic linkage of glycosides. Characterization of the beta‐glucosidase in G. mellonella could be a significant stage for a better comprehending of its role and establishing a safe and effective control procedure primarily against G. mellonella and also some other insect pests. Laboratory reared final instar stage larvae were randomly selected and homogenized for beta‐glucosidase activity assay and subsequent analysis. The enzyme was purified to apparent homogeneity by salting out with ammonium sulfate and using sepharose‐4B‐l ‐tyrosine‐1‐naphthylamine hydrophobic interaction chromatography. The purification was 58‐fold with an overall enzyme yield of 29%. The molecular mass of the protein was estimated as ca. 42 kDa. The purified beta‐glucosidase was effectively active on para/ortho‐nitrophenyl‐beta‐d ‐glucopyranosides (p‐/o‐NPG) with Km values of 0.37 and 1.9 mM and Vmax values of 625 and 189 U/mg, respectively. It also exhibits different levels of activity against para‐nitrophenyl‐β‐d ‐fucopyranoside (p‐NPF), para/ortho‐nitrophenyl β‐d ‐galactopyranosides (p‐/o‐NPGal) and p‐nitrophenyl 1‐thio‐β‐d ‐glucopyranoside. The enzyme was competitively inhibited by beta‐gluconolactone and also was very tolerant to glucose against p‐NPG as substrate. The Ki and IC₅₀ values of δ‐gluconolactone were determined as 0.021 and 0.08 mM while the enzyme was more tolerant to glucose inhibition with IC50 value of 213.13 mM for p‐NPG.     

Allele Frequency of VNTR Locus D1S80 Observed in Denizli Province of Turkey

The variable numbers of tandem repeats (VNTR) locus D1S80, located on chromosome 1 (1p35-36), has a repeat unit 16 bp in length, and different numbers of these repeat units have been observed for populations of different origins and ethnicity. We used a molecular identification method based on capillary electrophoresis separation to analyze D1S80 locus polymorphism among 74 subjects from Denizli province, Turkey, finding an amplified fragment length size of 379–635 bp. Allele repeat numbers were deduced from these sizes and sequence comparison. The most common alleles were repeat units 24 (34.3%) and 18 (22.4%), with frequencies of 0.414 and 0.207, respectively. Other alleles were 25 (7.86%), 28 (5.71%), 22 (4.25%), and 29 (2.86%). The allele with 23 repeat units was not observed. Results were in Hardy–Weinberg linkage disequilibrium. Observed heterozygosity was 0.614, and expected heterozygosity was 0.787. Theta(k) value was 4.86 (95% confidence interval limits). Capillary electrophoresis is a powerful approach for accurate identification of VNTR loci, especially for low base pair units like D1S80, for prenatal diagnosis, linkage analysis, forensic identification, paternity testing, anthropological research, and phylogenetic studies.     

Effect of gem 2,2′-disubstitution and base in the formation of spiro- and ansa-1,3-propandioxy derivatives of cyclotriphosphazenes

The gem-dialkyl effect has been investigated in the reactions of cyclotriphosphazene, N₃P₃Cl₆1, with various 2,2′-derivatives of 1,3-propandiol, CXY(CH₂OH)₂, in either THF or DCM to form spiro (6-membered) and ansa (8-membered ring) derivatives. The reactions were made with a number of symmetrically-substituted (X = Y, methyl, ethyl, n-butyl and a malonate ester) and unsymmetrically-substituted (X ≠ Y, methyl/H, phenyl/H, methyl/n-propyl, ethyl/n-butyl and Br/NO₂) 1,3-propandiols. The products were analysed by ¹H and ³¹P NMR spectroscopy and some of the spiro and ansa derivatives were also characterized by X-ray crystallography. Reactions of 1 with unsymmetrically-substituted 1,3-propandiols results in the formation of two structural isomers of ansa-substituted compounds, both isomers (endo and exo) have been structurally-characterized by X-ray crystallography for the ethyl/n-butyl derivative. It is found that the regioselectivity of the reaction is changed when the base is changed. The relative proportions of spiro and ansa compounds formed under different reaction conditions were quantified by ³¹P NMR measurements of the reaction mixtures. The results were rationalised mainly in terms of the electronic effect of the substituents, whereas the steric effect has a secondary role in the formation of both spiro and ansa compounds.