Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.     

Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2

The guanine nucleotide exchange factor Cdc24, the GTPase Cdc42, and the Cdc42 effectors Cla4 and Ste20, two p21-activated kinases, form a signal transduction cascade that promotes mitotic exit in yeast. We performed a genetic screen to identify components of this pathway. Two related bud cortex-associated Cdc42 effectors, Gic1 and Gic2, were obtained as factors that promoted mitotic exit independently of Ste20. The mitotic exit function of Gic1 was dependent on its activation by Cdc42 and on the release of Gic1 from the bud cortex. Gic proteins became essential for mitotic exit when activation of the mitotic exit network through Cdc5 polo kinase and the bud cortex protein Lte1 was impaired. The mitotic exit defect of cdc5-10 Deltalte1 Deltagic1 Deltagic2 cells was rescued by inactivation of the inhibiting Bfa1-Bub2 GTPase-activating protein. Moreover, Gic1 bound directly to Bub2 and prevented binding of the GTPase Tem1 to Bub2. We propose that in anaphase the Cdc42-regulated Gic proteins trigger mitotic exit by interfering with Bfa1-Bub2 GTPase-activating protein function.     

Determinants of 14-3-3σ Protein Dimerization and Function in Drug and Radiation Resistance

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the “undruggable” oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu¹² and Tyr⁸⁴ play important roles in 14-3-3σ dimerization, the non-core residue Phe²⁵ appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val⁸¹) is less important than Phe²⁵ in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu¹², Phe²⁵, or Tyr⁸⁴ dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G₂/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.     

Temperature responses of carbon mineralization in conifer forest soils from different regional climates incubated under standard laboratory conditions

¹⁴C‐labelled straw was mixed with soils collected from seven coniferous forests located on a climatic gradient in Western Europe ranging from boreal to Mediterranean conditions. The soils were incubated in the laboratory at 4°, 10°, 16°, 23° and 30 °C with constant moisture over 550 days. The temperature coefficient (Q₁₀) for straw carbon mineralization decreased with increasing incubation temperatures. This was a characteristic of all the soils with a difference of two Q₁₀ units between the 4–10° and the 23? 30 °C temperature ranges. It was also found that the magnitude of the temperature response function was related to the period of soil incubation. Initial temperature responses of microbial communities were different to those shown after a long period of laboratory incubation and may have reflected shifts in microbial species composition in response to changes in the temperature regime. The rapid exhaustion of the labile fractions of the decomposing material at higher temperatures could also lead to underestimation of the temperature sensitivity of soils unless estimated for carbon pools of similar qualities. Finally, the thermal optima for the organic soil horizons (Of and Oh) were lower than 30 °C even after 550 days of incubation. It was concluded that these responses could not be attributed to microbial physiological adaptations, but rather to the rates at which recalcitrant microbial secondary products were formed at higher temperatures. The implication of these variable temperature responses of soil materials is discussed in relation to modelling potential effects of global warming.     

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.     

柚木无性系愈伤组织诱导及植株再生

以柚木优良无性系71-14组培苗节间茎段为材料,MS 为基本培养基,采用正交设计对6-BA、IBA、TDZ、NAA 等4个生长调节剂各4水平进行愈伤组织诱导,并以最佳组合使用不同浓度的 TDZ 进行柚木愈伤组织再生.结果表明：TDZ 对形成具再生能力的致密型愈伤组织影响最大,低浓度水平的 TDZ 和6-BA 更易形成致密型愈伤组织;以愈伤组织大小、诱导率和致密型所占比例采用隶属函数法评定得出最优的愈伤组织诱导培养基为 MS＋0．9 mg·L-16-BA＋0．04 mg·L-1 IBA＋0．02 mg·L-1 TDZ＋0．8 mg·L-1 NAA,愈伤组织诱导率达80．78％、平均直径1．65 cm,获致密型愈伤组织83．0％;得出优化的再生培养基为 MS＋0．132 mg·L-1 TDZ,分化率为34．22％;初步建立了以茎段为外植体的柚木优良无性系71-14的再生体系,为柚木转基因技术的研究提供技术支撑.