Regulation of intracellular pH by Ca2+ - activated proton channel

Background and aim: We have characterized a membrane current associated with a decrease in pHi, which can be induced by either elevating intracellular calcium or extracellular application of methylmercury (a potent agent in elevating intracellular Ca²⁺ concentration) in the alveolar macrophages bathing in the impermeant bilateral cesium aspartate solution.

Results: Decreasing pHi and elevating [Ca²⁺]i profoundly enhanced, but H-7 (a broad-spectrum kinase inhibitor), W-7 (a selective calmodulin antagonist) and KN-93 (a calmodulin kinase II inhibitor) inhibited the currents.

Conclusion: These results indicate that rat alveolar macrophages possess a calcium-activated and pHi-sensitive proton channel which can be phosphorylated and activated by calmodulin kinase II.     

负荷渐增式训练对老年小鼠骨骼肌卫星细胞AMPK磷酸化的影响

目的 探讨负荷渐增式训练对老年小鼠骨骼肌卫星细胞腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）磷酸化的影响。方法 实验小鼠分为 3 组：青年对照组（YC组，n=12）、老年对照组（OC组，n=12）与老年运动组（OT组，n=12）。OT组进行负荷渐增式训练，流式细胞分选技术分离CD45-/CD31-/Sca1-/VCAM(CD106)+细胞群体，分选细胞通过desmin、Myod肌原性染色以及成肌分化诱导培养进行肌卫星细胞鉴定，免疫组化结合Western blotting方法检测肌卫星细胞p-AMPK水平。结果 YC组骨骼肌卫星细胞AMPK及p-AMPK表达水平显著高于OC组（P<0.05）；OT组与OC组AMPK表达无明显变化（Ｐ>0.05），而OT组p-AMPK表达水平显著高于OC组（P<0.05）。结论 负荷渐增式训练可促进老年小鼠骨骼肌卫星细胞AMPK磷酸化，改善老年小鼠骨骼肌能量代谢。     

Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

Currently, Stathmin1 (STMN1) and phospho-STMN1 levels in breast cancers and their clinical implications are unknown. We examined the expression of STMN1 and its serine phospho-site (Ser16, Ser25, Ser38, and Ser63) status by immunohistochemistry. Using Cox regression analysis, a STMN1 expression signature and phosphorylation profile plus clinicopathological characteristics (STMN1-E/P/C) was developed in the training set (n = 204) and applied to the validation set (n = 106). This tool enabled us to separate breast cancer patients into high- and low-risk groups with significantly different disease-free survival (DFS) rates (P < 0.001). Importantly, this STMN1-E/P/C model had a greater prognostic value than the traditional TNM classifier, especially in luminal subtype breast cancer (P = 0.002). Further analysis showed that patients in the low-risk group would benefit more from adjuvant paclitaxel-based chemotherapy (P = 0.002). In conclusion, the STMN1-E/P/C signature is a reliable prognostic indicator for luminal subtype breast cancer and may predict the therapeutic response to paclitaxel-based treatments, potentially facilitating individualized management.     

FoxO1转录因子及其与心血管疾病的研究进展

FoxO1转录因子是FoxO家族中起主要作用的成员，由FKHR基因编码，参与调节代谢、氧化应激反应、免疫稳态、细胞周期、细胞凋亡等过程，与心血管疾病的病理生理过程密切相关。翻译后修饰如磷酸化、乙酰化、泛素化等可以调节FoxO1的活性。本文将简要对FoxO1的基本结构和功能、活性的调节及其在心血管疾病中的研究进展进行综述。     

Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation

Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2A^B56 allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2A^B56 also stabilizes kinetochore–microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2–E3 interplay to coordinate ubiquitination with critical events during cell division.By promoting the ubiquitination and proteasomal degradation of anaphase inhibitors, the anaphase-promoting complex (APC/C) triggers sister chromatid separation and mitotic exit (1–5). The APC/C also targets kinases and microtubule-binding proteins that ensure accurate assembly of the mitotic spindle. Misregulation of the APC/C has dramatic consequences for cell cycle control; whereas APC/C inhibition causes mitotic arrest and cell death, its untimely activation results in aneuploidy, a common feature of human cancer cells (6).As a RING-dependent E3 ligase, the APC/C stimulates the transfer of ubiquitin from the catalytic cysteine of E2 conjugating enzymes to lysine residues in substrates. In most cases, the APC/C initiates chain formation by using a specific E2, Ube2C (7–10). Once the first ubiquitin molecules have been attached to substrates, another conserved E2, Ube2S, extends K11-linked chains that are recognized by the proteasome for degradation (11–16). Ube2S frequently acts on short chains rather than on single ubiquitin subunits, thereby producing branched conjugates that impart high affinity for proteasomal receptors (13). Consistent with an important role in cell division, activation of Ube2S during mitosis results in a dramatic increase in the abundance of K11 linkages (17, 18), a chain topology required for APC/C-dependent substrate degradation (19).As with many key cell cycle regulators, the APC/C and Ube2S need to be under tight control, and overexpression of Ube2S can promote tumor growth and metastasis in mice (20). The correct timing of APC/C activation is ensured by the spindle checkpoint, a signaling cascade turned on by kinetochores that have not achieved bipolar attachment to the spindle (4, 21, 22). Spindle checkpoint signaling leads to formation of the mitotic checkpoint complex (MCC), composed of Mad2, BubR1, Bub3, and Cdc20. When bound to the APC/C, the MCC competes for recognition of substrate KEN boxes and puts the APC/C coactivator Cdc20 in a position where it is unable to engage another degron, the D box (23–25). In contrast, the MCC does not occupy the binding sites for APC/C E2s or impede the ability of the APC/C to stimulate ubiquitin transfer by Ube2S (12). Thus, although overexpression of Ube2S has been associated with tumorigenesis, the mechanisms that restrict its activity during mitosis have remained elusive.RING-E3s, such as the APC/C, engage their E2 enzymes in a dynamic manner (26). On binding a charged E2, the RING domain stabilizes a closed conformation between the E2 and its donor ubiquitin (14, 27–30). Once this ubiquitin is transferred to a target lysine, the E2 dissociates from the RING domain to allow for its recharging by the E1 (31). For most RING-E3s, the cycles of E2 engagement and dissociation are thought to occur constitutively (32), and only a few examples of controlled E2 activation are known. Access of Cdc34 to its specific RING-E3, the Skp1-Cul1-F box (SCF) complex, can be regulated by phosphorylation or competition with the inhibitory protein glomulin (33, 34). Reminiscent of this situation, Ube2S interacts with the APC/C in a cell cycle-dependent manner, and depletion of Cdc20 prevents Ube2S from stably binding to the APC/C in cells (12, 15). However, as part of the MCC, Cdc20 already associates with the APC/C during prometaphase, when APC/C activity must be low to allow sufficient time for chromosome alignment. How the ability of Ube2S to build ubiquitin chains is restricted during early stages of mitosis to safeguard cells against premature APC/C activation remains unknown.In this study, we identified a mechanism that establishes how the RING-E3 APC/C activates Ube2S at the right time and place. In early mitosis, phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents the stable association of Ube2S with Cdc20 and the APC/C. Conversely, removal of the inhibitory mark on Cdc20 by the phosphatase PP2A^B56 allows Ube2S to engage the APC/C and catalyze ubiquitin chain elongation. PP2A^B56 also stabilizes the kinetochore–microtubule interface to silence the spindle checkpoint (35, 36), suggesting that cells regulate the interplay between RING-E3s and their E2s to coordinate ubiquitination with important events in cell division.