How do proteins in our body achieve muscle movement?

This poster provides an introduction into the studies of proteins, involved in muscle movement and contraction, that we are undertaking at the ANU's John Curtin School of Medical Research. We study interactions between these proteins on a molecular level, with the aim of better understanding of physiological processes that take place in our muscle cells under normal conditions and in disease states. The outcomes of our research might pave the way for the treatment of pathological conditions, associated with skeletal and cardiac muscle disorders.NHMRC APP112620

3D mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.The authors want to thank the Brigham and Women’s Hospital Biomedical Research Institute (to MS), the Australian National Health and the Medical Research Council (471418 to AD, MC and PB), and the European Commission (Marie Curie Action PIOF-GA-2009-237120 to AP-M)

Outcomes of self-control plans on acrylamide levels in processed food

In 2002, researchers from Stockholm University discovered the presence of acrylamide (AA) in processed foods. This substance has been classified as “probably carcinogenic to humans” by the International Agency for Research on Cancer. In response to the alarming finding, the European Commission issued recommendations (2004/394/EC, 2010/307/EU, and 2013/647/EU), guiding food business operators, raising awareness, and promoting good manufacturing practices to minimize AA formation. These efforts laid the foundation for the comprehensive measures in Regulation (EU) 2017/2158. The Regulation implemented specific measures during production to reduce the amount of AA in food. This study monitored the AA levels in 15,674 samples from 12 processed food commodities. Potato-based products and coffee were found to be the main sources of AA exposure. The “baby foods” and “soft bread” food categories had the lowest contamination levels. The data were then compared to the information previously published by the European Food Safety Authority to assess the trend over time and the effectiveness of the mitigation measures. The results showed a decrease in AA contamination levels for most food categories, particularly for baby foods

A Structural Basis for Cellular Uptake of GST-Fold Proteins

It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.This work was supported by Grant DP0558315 Australian Research Council (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cyclization of the intrinsically disordered α 1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α1S subunit of the dihydropyridine receptor (DHPR). The II-III loop has been pre

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca2+ release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca2+ release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use