Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage.

Stroke prognosis is negatively associated with an elevation of serum bilirubin, but how bilirubin worsens outcomes remains mysterious. We report that post-, but not pre-, stroke bilirubin levels among inpatients scale with infarct volume. In mouse models, bilirubin increases neuronal excitability and ischemic infarct, whereas ischemic insults induce the release of endogenous bilirubin, all of which are attenuated by knockout of the TRPM2 channel or its antagonist A23. Independent of canonical TRPM2 intracellular agonists, bilirubin and its metabolic derivatives gate the channel opening, whereas A23 antagonizes it by binding to the same cavity. Knocking in a loss of binding point mutation for bilirubin, TRPM2-D1066A, effectively antagonizes ischemic neurotoxicity in mice. These findings suggest a vicious cycle of stroke injury in which initial ischemic insults trigger the release of endogenous bilirubin from injured cells, which potentially acts as a volume neurotransmitter to activate TRPM2 channels, aggravating Ca2+-dependent brain injury.

Effects of calcium-binding sites in the S2-S3 loop on human and Nematostella vectensis TRPM2 channel gating processes.

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.

The far-upstream element-binding protein 2 is correlated with proliferation and doxorubicin resistance in human breast cancer cell lines.

Far-upstream element (FUSE)-binding protein 2 (FBP2) was a member of single-stranded DNA-binding protein family; it played an important role in regulating transcription and post-transcription and is involved in the regulation of C-MYC gene expression in liver tumors. However, the role of FBP2 in breast cancer and its mechanism has not been studied yet. Here, we discovered that FBP2 was up-regulated in breast cancer tissues and breast cancer cell lines. Moreover, immunohistochemistry analysis demonstrated that up-regulated FBP2 was highly associated with tumor grade, Ki-67, and poor prognosis, which was an independent prognostic factor for survival of breast cancer patients. At the cellular level, we found that FBP2 was correlated with cell cycle progression by accelerating G1/S transition, and knockdown of FBP2 could weaken cell proliferation, anchorage-independent cell growth, while enhancing the sensitivity of breast cancer cells to doxorubicin. More importantly, we found that activation of PI3K/AKT pathway could phosphorylate FBP2, and then make FBP2 shuttle from cytoplasm into the nucleus, which was the main mechanism of breast cancer cell proliferation and drug resistance. Taken together, our findings supported the notion that FBP2 might via PI3K/AKT pathway influence breast cancer progression and drug resistance, which might provide a new target for the design of anti-cancer drugs for breast cancer patients.

Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells.

Adenylate cyclase-associated protein 1 (CAP1) is a conserved protein that was found to be up-regulated in breast cancer and related to the migration of breast cancer. We verified its roles in breast cancer specimens and cell lines. In our results, 71 of 100 specimens of breast cancer showed high levels of CAP1 by immunohistochemistry. Associated with statistical analysis, we saw that CAP1 was related to the grade of breast cancer. In MDA-MB-231, the expression of CAP1 was the highest and by knocking down the expression of CAP1 in MDA-MB-231, its ability for proliferating and migrating apparently decreased and induced changes in morphology, which were related to the arrangement of F-actin. Therefore, CAP1 might be a potential molecular targeted therapy for surgery and immune treatment.