Identification and characterization of the localization and expression of CLIC4 under redox conditions in mammalian cells

Chloride intracellular ion channel-4 (CLIC4) is a member of the CLIC family of proteins which were originally identified as channels of intracellular membranes permeable to ion chloride ions. Its expression and localization to intracellular membrane is sensitive to oxidative stress. This sensitivity of CLIC4 is indicated with its physiological redox regulatory function either as an oxidoreductase enzyme or an ion channel in response to decrease the cellular glutathione level, ROS accumulation, and consequently non-native disulfide formation in the cells. The pathways for disulfide formation are well characterized. However, the understanding of redox state of CLIC4 and possibility to participate in reductive pathway to removing the non-native disulfide bond is still limited and whether CLIC4 as a membranebinding protein with oxidoreductase activity might be needed in the reduction pathway either in the cytosol or ER, are the questions we need to address. In this project the oxidative stress induced by TNF-α and CLIC4 in response to TNF-α can be re-localized to the ER from the cytosol. The ER is a host for disulfide formation within folding proteins entering the mammalian secretory pathway. The consequence of oxidative stress in the ER is the accumulation of misfolded and unfolded proteins. The mammalian cells have a family of oxidoreductase that is thought to be isomerised non-native disulfide bonds. This reductive catalytic activity of oxidoreductases is maintained via a reductive pathway. For CLIC4 to act as an oxidoreductase for performing isomerisation or reduction reactions, it must be preserved in a reduced position. Here, by mass spectrometry, using purified proteins and ER microsomal membrane following TNF-α induced oxidative stress, we illustrate CLIC4 is predominantly placed in a reduced state in the intact cells, demonstrating a reductive pathway is prepared in mammalian cells and CLIC4 can be involved with this pathway as an oxidoreductase through its either enzyme catalytic activity or ion channel activity. In this project, the glutathione has identified to be responsible for the reduction of CLIC4 during oxidative stress. Furthermore, when inhibitors of glutathione synthesis or reductase are added to the cells, CLIC4 is not reduced. The results demonstrate that glutathione plays a direct role in the isomerisation of disulfide bonds by maintaining CLIC4 in a reduced state. To confirm the reduction effect of GSH on CLIC4 and overall microsomal membrane protein in response to TNF-α, we have applied a cysteine-reactive tandem mass tag (Iodo-TMT) to differentially label cysteine residues and analyse the overall protein expression level and redox state into one-step analysis. The individually labeled samples have been pooled in differential combinations to create multiple six-plex samples to determine the effect of GSH on cysteine oxidation and overall protein expression in the microsomal membrane. The result highlights the redox status of CLIC4 under reduction of GSH was confirmed with MS/LC, and the TMT-labeling detected the redox state of the overall microsomal membrane proteins with respect to cysteine oxidation and protein expression. This study is important because CLIC4 as either a membrane binding protein or an ion channel can be considered as a mis-component in reductive pathway

Comparison the diagnostic value of serological and molecular methods for screening and detecting Chlamydia trachomatis in semen of infertile men: A cross-sectional study

Background: Chlamydia trachomatis (CT) with damaging effects on sperm quality parameters can often cause infertility in men. Objective: The main objective of this study was to determine the diagnostic value of polymerase chain reaction (PCR) and enzyme linked immuno sorbent assay (ELISA) for screening and detecting CT in semen samples of infertile men. Materials and Methods: In this cross-sectional study, 465 men referring to the clinical laboratory of Royan Institute were chosen for primary screening and detection of the presence of CT. 93 samples were normozoospermia with normal sperm parameters i.e. sperm number, motility and morphology (Asymptomatic) and 372 had abnormal sperm parameters (Symptomatic) in semen analysis. ELISA test was performed as the screening test. Samples with optical density (OD) >0.200 were selected as the case and asymptomatic samples with OD 0.400 as the ELISA positive, the diagnostic value of CT-ELISA positive in symptomatic and asymptomatic infertile patients were 0.019 (7 of 372) and 0.021 (2 of 93), respectively. There was no relationship between the presence of CT infection and different sperm abnormalities. Conclusion: The anti-CT IgA ELISA test may be introduced as an appropriate tool for screening purpose in the seminal plasma to select suspicious samples for PCR confirmatory tests

The expression of Cysteine-Rich Secretory Protein 2 (CRISP2) and miR-582-5p in seminal plasma fluid and spermatozoa of infertile men

Cysteine-Rich Secretory Protein 2 (CRISP2) plays an important role in the morphology and motion of male ejaculated spermatozoa. The association of its expression with some miRNAs is also well known. The aim of this study was to determine the expression of CRISP2 and mir-582 in the seminal plasma fluid and spermatozoa of three groups of infertile men and the possible association of their expressions. In this experimental study, the expression of CRISP2 in seminal plasma fluid and spermatozoa of 17 men with asthenozoospermia, 15 men with teratozoospermia, 17 men with teratoasthenozoospermia, and 18 infertile individuals with normozoospermia were measured using western blotting. Then by using bioinformatics studies, miR-582-5p was nominated as a CRISP2-associated miRNA, and its expression was evaluated by means of Real-Time PCR. Comparison of expression of CRISP2 and miRNA-582 in the studied groups was analyzed by t-test and Mann-Whitney U test. The expression of CRISP2 showed a significant reduction in the spermatozoa and seminal plasma fluid of all three groups, (p < 0.05). MiR-582-5p expression significantly increased in teratozoospermia patients (<0.05), and significantly decreased in teratoasthenozoospermia patients (p < 0.05). Meanwhile, changes in the expression of miR-582-5p in teratoasthenozoospermia individuals was associated with a decrease in the expression of CRISP2, which could represent the potential role of miR-582-5p in regulation of CRISP2 expression in teratoasthenozoospermia individuals