Biochemical characterisation of the enzymatic activity of chloride intracellular ion channel proteins

University of Technology Sydney. Faculty of Science.The chloride intracellular ion channel (CLIC) family, are the group of unusual proteins that exist either in monomeric soluble or integral membrane form. This family is comprised of six protein members in humans, CLIC1-CLIC6. CLIC1 is classified as a “metamorphic” protein, which means it can exist in two stable tertiary conformations. The CLIC proteins can spontaneously insert into phospholipid membranes from their soluble state where they act as ion channel proteins. In addition, the CLIC proteins have structural similarities with both the Glutathione-S-Transferases and glutaredoxin enzyme families. Recently it has been demonstrated by in vitro assay systems that the monomeric CLIC proteins have similar “oxidoreductase” activity to the Glutaredoxin family. Therefore, following on from these new discoveries, further detailed characterization of their enzymatic activity was required, which was the main purpose of this research project. The first objective of this thesis was to determine the important structural regions of CLICs that could be sensitive to the various environmental conditions. In that regards, a comparative study was conducted on both “Histidine tagged” and “non-tagged” CLIC proteins in order to check whether the “6 Polyhistidine tag” and “imidazole” compound that are routinely used in the preparation and purification of the recombinant CLIC proteins, interferes with the functional activity of CLIC1 and CLIC3 proteins. Indeed, the results indicate that the His tag altered the enzymatic function by lowering its catalytic activity. In addition, the imidazole compound was also found to interfere with CLIC’s catalytic activity by acting as a second substrate that led to inaccurate assay measurements. As such, it is recommended that removal of both the “His-tag” and “imidazole” be done in order to avoid any interference in subsequent enzyme characterization studies. The project then proceeded to determine the optimal conditions for the enzymatic activity of the CLIC1 and 3 enzymes, across a range of pH and temperatures. CLIC3 was found to be heat resistant compared to CLIC1, which demonstrated heat sensitivity at higher temperatures. CLIC1 was seen to decrease in it activity and stability at increased temperature. The optimal thermal activity of both proteins obtained was 37°C in the HEDS enzyme assay. However, these studies revealed that the optimal catalytic activity of CLIC3 is obtained under acidic conditions (around pH 6), in contrast to CLIC1 that was optimally active at under more physiological conditions (pH 7). Furthermore, this study focused on exploring alternate new substrates for CLIC enzymes. It was found that soluble CLIC3 and CLIC1, demonstrate an affinity to a number of substrates including Imidazole, DHA and sodium selenite. The inhibitory effect of the ion channel blocker, drug IAA94 on the oxidoreductase activity of these two proteins was also examined, which showed that it inhibited the enzymatic activity of CLIC3 similar to CLIC1. The current study also sought to define the kinetic profile of oxidoreductase catalytic activity of these CLIC proteins. Based on our characterization study, kinetic constants (Vmax, Km) for both CLIC1 and CLIC3 were determined. By comparing these catalytic efficiencies, it was evident that both CLIC1 and CLIC3 obey a Michaelis-Menten kinetics module with Vmax values of (2.026, 3.33 μM/min) and Km value of (2.503, 0.9941 mM) respectively. Also, this characteristic study revealed that both CLIC1 and CLIC3 enzymes are following first-order kinetic reactions with a Vmax value of (1.4mM/min) and (0.7mM/min), respectively. These combined in vitro studies revealed the distinct enzymatic activity and profile of each protein member. Such information is critical in beginning to unravel the newly described enzymatic activity of these proteins and will allow the discrete roles within cells to be assigned to each of these proteins. Future studies determining intracellular function will need to take into account the role of pH and local environmental conditions, along with consideration of relevant substrates and likely protein targets for the enzymatic oxidoreductase activity of the CLIC proteins

<i>In Vitro</i> Enzymatic Studies Reveal pH and Temperature Sensitive Properties of the CLIC Proteins

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins’ enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment

Cost-Effectiveness of Cardiac Biomarkers as Screening Test in Acute Chest Pain

Introduction: Acute chest pain is an important and frequently occurring symptom in patients. Chest pain is often a sign of ischemic heart disease. Associated findings of electrocardiograph (ECG) are rather heterogeneous, and traditional cardiac biomarkers such as Creatine Kinase-MB (CK-MB) suffer from low cardiac specificity and sensitivity. In this study cost effectiveness of cardiac biomarkers single quantitative measurement was examined.Methods: The present descriptive-analytic study conducted on patients who were asked for troponin I and CK-MB. All patients who referred to Emergency unit of Tabriz Imam Reza educational-medical center during January 2012 to July the 2013 were included in study. All patients included in the study were documented in terms of age, sex, working shift of referring, main complaint of patient, symptoms in referring, ECG findings, and results of troponin I and CK-MB tests.Results: In this study, 2900 patients were studied including 1440 (49.7%) males and 1460 (50.3%) females. Mean age of patients was 62.91 (SD=14.36). Of all patients 1880 (64.8%) of patients referred during 8 a.m. to 8 p.m. and 1020 (35.2%) patients were referred during 8 p.m. to 8 a.m. The sensitivity of cardiac biomarkers’ test in diagnosing Acute Coronary Syndrome (ACS) disease was calculated as 44.8% and its specificity was 86.6%. For diagnosing Acute Myocardial Infarction (AMI), sensitivity of cardiac biomarkers’ test was 72.2% and its specificity was 86%. None of patients who were finally underwent unstable angina diagnosis showed increase in cardiac enzymes.Conclusion: In conclusion, cardiac biomarkers can be used for screening acute chest pains, also cost effectiveness of cardiac biomarkers, appropriate specificity and sensitivity can guarantee their usefulness in emergency room