Chronic stress inhibits testosterone synthesis in Leydig cells through mitochondrial damage via Atp5a1.

Stress is one of the leading causes of male infertility, but its exact function in testosterone synthesis has scarcely been reported. We found that adult male rats show a decrease in bodyweight, genital index and serum testosterone level after continual chronic stress for 21 days. Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS analysis identified 10 differentially expressed proteins in stressed rats compared with controls. A strong protein interaction network was found to be centred on Atp5a1 among these proteins. Atp5a1 expression significantly decreased in Leydig cells after chronic stress. Transfection of Atp5a1 siRNAs decreased StAR, CYP11A1, and 17ß-HSD expression by damaging the structure of mitochondria in TM3 cells. This study confirmed that chronic stress plays an important role in testosterone synthesis by regulating Atp5a1 expression in Leydig cells.

Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins represents a unique membrane protein family that facilitates the insertion, folding, and assembly of a cohort of α-helical membrane proteins in all kingdoms of life, yet its underlying mechanisms remain elusive. We report the crystal structures of the full-length Thermotoga maritima YidC (TmYidC) and the TmYidC periplasmic domain (TmPD) at a resolution of 3.8 and 2.5 Å, respectively. The crystal structure of TmPD reveals a ß-supersandwich fold but with apparently shortened ß strands and different connectivity, as compared to the Escherichia coli YidC (EcYidC) periplasmic domain (EcPD). TmYidC in a detergent-solubilized state also adopts a monomeric form and its conserved core domain, which consists of 2 loosely associated α-helical bundles, assemble a fold similar to that of the other YidC homologues, yet distinct from that of the archaeal YidC-like DUF106 protein. Functional analysis using in vivo photo-crosslinking experiments demonstrates that Pf3 coat protein, a Sec-independent YidC substrate, exits to the lipid bilayer laterally via one of the 2 α-helical bundle interfaces: TM3-TM5. Engineered intramolecular disulfide bonds in TmYidC, in combination with complementation assays, suggest that significant rearrangement of the 2 α-helical bundles at the top of the hydrophilic groove is critical for TmYidC function. These experiments provide a more detailed mechanical insight into YidC-mediated membrane protein biogenesis.-Xin, Y., Zhao, Y., Zheng, J., Zhou, H., Zhang, X. C., Tian, C., Huang, Y. Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

Cell apoptosis in the testis of male rats is elevated by intervention with ß-endorphin and the mu opioid receptor.

ß-endorphin (ß-EP) is involved in the regulation of male germ cells; however, little is known about the effect of ß-EP on primary germ cells via opioid receptors. In this study, we first revealed significant cell apoptosis in the testis of male rats after ß-EP intervention. Subsequently, the expression of the mu opioid receptor (MOR) was detected in both Leydig cells (LCs) and spermatogonia (SGs) by fluorescence colocalization; overlapping signals were also detected in apoptotic cells. In addition, LCs and SGs were separated from the testis of male rats and primary cells were treated with ß-EP; this increased the mRNA levels of MOR and was accompanied by acute cell apoptosis. Our findings provide a foundation for the further study of apoptosis in reproductive cells regulated by ß-EP and the MOR receptor.

ML365 inhibits lipopolysaccharide-induced inflammatory responses via the NF-κB signaling pathway.

ML365 is a selective inhibitor of the twik-related acid-sensitive potassium channel 1/two-pore domain channel subfamily k member 3 two-pore domain potassium channel. There are no functional studies of the relationship between ML365 and inhibition of inflammation. In this study, we evaluated the anti-inflammatory effect of ML365 on lipopolysaccharide (LPS)-induced inflammation and elucidated the possible mechanism. ML365 showed no cytotoxicity and did not induce apoptosis on RAW264.7 cells and inhibited nitric oxide production. ML365 suppressed the release of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1ß measured using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction assays. LPS-induced activation and co-localization of NF-κB was inhibited by ML365 pre-treatment. ML365 inhibited the protein expression of Erk, p38 and Jnk. In vivo, ML365 appeared to prevent pathological damages in the LPS-induced endotoxin shock model. These findings suggest that ML365 inhibits LPS-induced inflammatory responses by regulating the NF-κB signaling pathway.

CW-EPR studies revealed different motional properties and oligomeric states of the integrin ß1a transmembrane domain in detergent micelles or liposomes.

Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or ß subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin ß1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin ß1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin ß1a-TM regions with different motional properties in micelles and a non-continuous integrin ß1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin ß1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin ß1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin ß1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes.