Development of covalent antagonists for β1- and β2-adrenergic receptors

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β₂-adrenergic receptor (β₂AR^H2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β₁- and β₂ receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β₁AR^I2.64C and β₂AR^H2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β₂AR^H2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β₂AR^H2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β₁AR^I2.64C and β₂AR^H2.64C mutant while retaining a 41-fold selectivity for the β₁AR wild type over β₂AR. These compounds may serve as valuable molecular tools for studying β₁/β₂ subtype selectivity or investigations on GPCR trafficking and dimerization.     

Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.     

Covalent stabilization of a small molecule-RNA complex

We demonstrate covalent bond formation between an RNA aptamer containing a cysteamine-tethered nucleobase and helix-threading peptides (HTPs) containing α-bromoacetamide N-termini. The reaction is high yielding and inhibited by a DNA strand Watson-Crick complementary to the aptamer sequence indicating covalent reaction is dependent on the high affinity HTP-binding site present in the folded aptamer. These results are important for future structural studies of HTP-RNA complexes and methods for the discovery of new high affinity analogs via covalent tethering strategies.     

Using molecular tethering to analyze the role of nuclear compartmentalization in the regulation of mammalian gene activity

The mammalian nucleus has a complex structural organization that dynamically interacts with the genome. Chromatin is organized into discrete domains by association with distinct nuclear compartments enriched in structural and regulatory proteins. Growing evidence suggests that gene activity is modulated by interactions with these sub-nuclear compartments. Therefore, analyzing how nuclear architecture controls genome activity will be necessary to fully understand complex biological processes such as development and disease. In this article we describe a molecular methodology involving inducible tethering that can be used to position genes at the inner nuclear membrane (INM)-lamina compartment. The consequences of such directed re-positioning on gene activity or other DNA transactions can then be analyzed. This approach can be generalized and extended to position genes or chromosomal domains within other nuclear compartments thereby greatly facilitating the analysis of nuclear structure and its impact on genome activity.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

The biology and polymer physics underlying large‐scale chromosome organization

Chromosome large‐scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high‐resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome.      

Predator–prey interactions in a restored eelgrass ecosystem: strategies for maximizing success of reintroduced bay scallops (Argopecten irradians)

Predation is a key determinant of community structure and function, and thus should play a central role in successful ecological restoration strategies. The bay scallop, Argopecten irradians, was once abundant in the coastal bays of Virginia, U.S.A., until the complete loss of their eelgrass habitat, Zostera marina, in the 1930s. With the successful restoration of Z. marina in these coastal bays, attention has turned to reintroducing A. irradians with the intent of producing a self‐sustaining population. The success of this effort requires an understanding of the sources and degree of natural mortality that A. irradians experiences throughout their ontogeny. The objectives of this study were to: (1) quantify predatory mortality during two successive life history stages of A. irradians, in both spring and fall spawns and (2) identify possible predators of A. irradians in the Virginia coastal bays. We conducted tethering experiments to quantify the proportional losses due to predation, and used otter trawls and suction samples to characterize the predator community over two consecutive years. Losses due to predation ranged from 4 to 80% per day, with smaller juveniles (<15 mm shell height) experiencing greater mortality in 2013, and larger juveniles (>20 mm shell height) in 2014, which we infer is driven by the absence and presence of adult blue crabs in 2013 and 2014, respectively. We propose that managers should look toward relatively inexpensive predator surveys to best judge both when and at what size restored species should be introduced into the wild.     

A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.     

Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations

Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET‐specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET‐specific subunits caused fragmentation of APPL1‐positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET‐specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.      

Fluorescence Lifetime Imaging of Interactions between Golgi Tethering Factors and Small GTPases in Plants

Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta . Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1^b, AtRab-H1^c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.