Effects of Tropomodulin 2 on Dendritic Spine Reorganization and Dynamics.

Dendritic spines are actin-rich protrusions that receive a signal from the axon at the synapse. Remodeling of cytoskeletal actin is tightly connected to dendritic spine morphology-mediated synaptic plasticity of the neuron. Remodeling of cytoskeletal actin is required for the formation, development, maturation, and reorganization of dendritic spines. Actin filaments are highly dynamic structures with slow-growing/pointed and fast-growing/barbed ends. Very few studies have been conducted on the role of pointed-end binding proteins in the regulation of dendritic spine morphology. In this study, we evaluated the role played by tropomodulin 2 (Tmod2)-a brain-specific isoform, on the dendritic spine re-organization. Tmod2 regulates actin nucleation and polymerization by binding to the pointed end via actin and tropomyosin (Tpm) binding sites. We studied the effects of Tmod2 overexpression in primary hippocampal neurons on spine morphology using confocal microscopy and image analysis. Tmod2 overexpression decreased the spine number and increased spine length. Destroying Tpm-binding ability increased the number of shaft synapses and thin spine motility. Eliminating the actin-binding abilities of Tmod2 increased the number of mushroom spines. Tpm-mediated pointed-end binding decreased F-actin depolymerization, which may positively affect spine stabilization; the nucleation ability of Tmod2 appeared to increase shaft synapses.

Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin.

Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca2+-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.164-154 and Tpm3.1265-155, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.1265-155R91P and Tpm1.164-154I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.

Structural studies of Myceliophthora Thermophila Laccase in the presence of deep eutectic solvents.

In this work, we elucidated the interactions between Myceliophthora thermophila laccase and deep eutectic solvent (DES) by crystallographic and kinetics analyses. Four types of DESs with different hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD), including lactic acid: betaine, glycerol: choline chloride, lactic acid: choline chloride and glycerol: betaine was used. The results revealed that different DES have different effects on laccase activity. Lactic acid-betaine (2:1) DES has shown to enhance laccase activity up to 300 % at a concentration ranged from 2% to 8% v/v, while glycerol: choline chloride and lactic acid: choline chloride DES choline chloride-based DES have found to possess inhibitory effects on laccase under the same concentration range. Detailed kinetic study showed that glycerol: choline chloride DES is a S-parabolic-I-parabolic mixed non-competitive inhibitor, where conformational changes can occur. The crystal structures of laccase with lactic acid: choline chloride DES (LCDES) were obtained at 1.6 Å. Crystallographic analysis suggested that the addition of LCDES causes changes in the laccase active site, but the increase in water molecules observed in the resulting crystal prevented laccase from experiencing drastic structural change. Fluorescence and circular dichroism spectroscopies were also applied to determine the effects of DES on the structural conformation of laccase. The results have confirmed that the presence of DES can trigger changes in the local environments of the amino acids in the active site of laccase which contributes to the changes in its activity and stability.

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments.

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1-Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three-dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin-binding sites of tropomodulin and generated a three-dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self-consistent three-dimensional model of tropomodulin assembly at the pointed end. The model of the pointed-end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.