Ectopic BH3-only protein Bim acts as a cochaperone to positively regulate Hsp70 in yeast.

The chaperone heat shock protein 70 (Hsp70) is conserved from bacteria to humans and is crucial for avoiding protein misfolding under stress. Bim functions, mainly as one of the B-cell lymphoma 2 (Bcl-2) family proapoptotic members, were identified to be a cochaperone of Hsp70. Herein, we reported that ectopic Bim could constitute the interactions with intrinsic Hsp70 and translate its positive cochaperone activity in vitro to the yeast growth promotion and help Hsp70 to fold its client Ras-like protein. With the help of a specific Hsp70/Bim disruptor, we illustrated that Hsp70/Bim dimers rescue yeast from heat shock. In an organism lacks apoptotic Bcl-2 factors, the proapoptotic Bim in mammalian cells exhibits prosurvival functions.

A "Three-in-One" Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ.

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN3 -OPD-Alk) by a "three-in-one" molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN3 -OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.

Targeting the Allosteric Pathway That Interconnects the Core-Functional Scaffold and the Distal Phosphorylation Sites for Specific Dephosphorylation of Bcl-2.

Protein phosphorylation is the most significant post-translational modification for regulating cellular activities, but site-specific modulation of phosphorylation is still challenging. Using three-dimensional NMR spectra, molecular dynamics simulations, and alanine mutations, we identified that the interaction network between pT69/pS70 and R106/R109 residues prevents the phosphorylation sites from exposure to phosphatase and subsequent dephosphorylation. A Bcl-2-dephosphorylation probe, S1-6e, was designed by installing a carboxylic acid group to a Bcl-2 inhibitor. The carboxyl group competitively disrupts the interaction network between R106/R109 and pT69/pS70 and subsequently facilitates Bcl-2 dephosphorylation in living cells. As a result, S1-6e manifests a more effective apoptosis induction in pBcl-2-dependent cancer cells than other inhibitors exhibiting a similar binding affinity for Bcl-2. We believe that targeting the allosteric pathways interconnecting the core-functional domain and the phosphorylation site can be a general strategy for a rational design of site-specific dephosphorylating probes, since the allosteric pathway has been discovered in a variety of proteins.

The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients.

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Proteolysis Targeting Chimeras for the Selective Degradation of Mcl-1/Bcl-2 Derived from Nonselective Target Binding Ligands.

Proteolysis targeting chimera (PROTAC) recruits an E3 ligase to a target protein to induce its ubiquitination and subsequent degradation. We reported success in the development of two PROTACs (C3 and C5) that potently and selectively induce the degradation of Mcl-1 and Bcl-2 (DC50 = 0.7 and 3.0 µM), respectively, by introducing the E3 ligase cereblon-binding ligand pomalidomide to Mcl-1/Bcl-2 dual inhibitors S1-6 and Nap-1 with micromolar-range affinity. C3-induced Mcl-1 ubiquitination translated into much more lethality in Mcl-1-dependent H23 cells than the most potent Mcl-1 occupancy-based inhibitor A-1210477 with nanomolar-range affinity. Moreover, structure-activity relationship analysis and molecular dynamic simulations discovered the structural basis for turning nonselective or promiscuous Bcl-2 family ligands into selective PROTACs. C3 and C5 exhibited reversible depletion in living cells, which provides a new potent toolkit for gain-of-function studies to probe the dynamic roles of Bcl-2 and Mcl-1 in apoptosis networks.

Antioxidant activity of phenylethanoid glycosides on glutamate-induced neurotoxicity.

Exposure of PC12 cells to 10 mM glutamate caused significant viability loss, cell apoptosis, decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA). In parallel, glutamate significantly increased the intracellular levels of ROS and intracellular calcium. However, pretreatment of the cells with acteoside and isoacteoside significantly suppressed glutamate-induced cellular events. Moreover, acteoside and isoacteoside reduced the glutamate-induced increase of caspase-3 activity and also ameliorated the glutamate-induced Bcl-2/Bax ratio reduction in PC12 cells. Furthermore, acteoside and isoacteoside significantly inhibited glutamate-induced DNA damage. In the mouse model, acteoside significantly attenuated cognitive deficits in the Y maze test and attenuated neuronal damage of the hippocampal CA1 regions induced by glutamate. These data indicated that acteoside and isoacteoside play neuroprotective effects through anti-oxidative stress, anti-apoptosis, and maintenance of steady intracellular calcium.

Protective role of phenylethanoid glycosides, Torenoside B and Savatiside A, in Alzheimer's disease.

The current study assessed the efficacy of two phenylethanoid glycosides (PhGs), Torenoside B (TB) and Savatiside A (SA), in the treatment of Alzheimer's disease (AD). The effects of TB and SA compounds were first assessed following amyloid beta (Aß)25-35 induction in SH-SY5Y cells at a range of concentrations. Their effects on cell viability and reactive oxygen species (ROS) were determined by performing MTT and dichlorofluorescin diacetate assays, respectively. The concentration of intracellular Ca2+ was determined using Fluo-3AM to stain SH-SY5Y cells. SA and TB treatments were also assessed in Aß25-35-induced mice. Y-maze and Morris water maze methods were utilized to assess murine learning and memory capability. The pathological changes of murine hippocampi was determined using H&E and Nissl staining. In addition, biochemical parameters associated with intracellular reactive oxygen pathways including Maleic dialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), acetylcholinesterase (AChE) and Calnexin were also assessed. TB and SA treatment in Aß25-35-induced SH-SY5Y cells resulted in the restoration of cell morphology, an increase of SOD and GSH-Px activity, a decrease in ROS, Ca2+ and MDA content, and a decrease in Calnexin expression. Furthermore, SA or TB treatment administered to Aß25-35-induced mice improved their spatial/non-spatial learning and memory capabilities. The efficacy of treatment was also supported by a marked change in the morphological structure of pyramidal neurons in the CA1 areas of murine hippocampi, as well as an increase of SOD and GSH-Px activity. Treatment also resulted in a decrease in MDA content, AchE activity and Calnexin expression in murine hippocampal tissue. As potential AD treatment drugs, SA and TB compounds have been demonstrated to alleviate the oxidative stress induced by Aß25-35 via the regulation of intracellular calcium homeostasis and Calnexin, preventing AD development.

Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein.

BACKGROUND: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. OBJECTIVE: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein. METHODS: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2. RESULTS: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site. CONCLUSION: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

The StLAC2 gene is required for cell wall integrity, DHN-melanin synthesis and the pathogenicity of Setosphaeria turcica.

Laccases are blue multicopper oxidases, play important roles in various biological processes. These processes include fungal dihydroxynaphthalene (DHN)-melanin biosynthesis and pathogenicity, cellular growth, morphogenesis, and differentiation. This study investigated functions of the laccase gene StLAC2 in Setosphaeria turcica. The Δlac2 mutant colony color was distinct from that of the S. turcica wild-type (WT) isolate, and the mutants exhibited defective conidial formation. In contrast to the WT, the mutants exhibited a lighter color on the 2, 2-azino-di-[3-ethylbenzo-thia-zolin-sulphonate] (ABTS) plates, and the intracellular laccase activity was lower. Notably, StLAC2 gene loss correlated with decreased DHN-melanin biosynthesis and affected the integrity of the cell wall, where the StLAC2 gene mutants showed thinner, more transparent walls with a higher number of mitochondria than the WT. The Δlac2 mutants also lost their pathogenicity in maize. The results indicated that the StLAC2 gene involved in cell wall integrity, melanin biosynthesis and appressorial and conidial formation.

[Snacks consumption among residents in Shenzhen City].

OBJECTIVE: To describe the status of snacks consumption among residents in Shenzhen. METHODS: By a multiple stage probability proportionate to size sampling, 12 communities were randomly selected from 8 districts of Shenzhen based on population proportion. In the second stage, 30 households were randomly selected from each community. In each household, 2 years or older were invited to take dietary survey. RESULTS: There were 66.1% residents consuming snacks. More girls ate snacks than boys (chi2 = 11.552, P < 0.01) and more children and adolescents ate snacks than adults (chi2 = 27.207, P < 0.01). The average daily intake of energy, protein, fat, carbohydrate, fiber, vitamin A, vitamin C, vitamin E, calcium, sodium,magnesium, iron and zinc from snacks were 107.8 kcal (451.5 kJ), 1.7 g, 0.8 g, 22.0 g, 1.1 g, 23.1 microg, 8.3 mg, 1.1 mg,17.0 mg, 9.3 mg, 21.0 mg, 0.8 mg and 0.4 mg. Food categories the most frequently consumed as snacks were fruit, pastry, milk and products, beverages and grains. CONCLUSION: It's important to strengthen the diet education among residents in Shenzhen, especially the knowledge how to select snacks correctly and rationally.

[The association between eating out of home and overweight/obesity among Chinese adults].

OBJECTIVE: To investigate the association between out-of-home eating (OH eating) and overweight/obesity among adults in China. METHODS: A total of 33 828 subjects aged 18-60 years old from the 2002 China National Nutrition and Health Survey were selected to calculate their daily consumption of food and nutrition, when people eating at home or out-of-home. The 24-hour dietary recall method for 3 consecutive days was used to collect food intake information ( not including the condiment intake). The logistic regression method was used to analyze the relationship between OH eating and overweight/obesity. RESULTS: The prevalence of OH eating among Chinese adults aged 18-60 years old was 28.3% (9 562/33 828) in 2002. Overall, the prevalence of OH eating was significantly greater among men compared to women (P < 0.05) , and the rate for men was 32.4% (5 117/15 805), and the rate for women was 24.7% (4 445/18 023). Besides, men who ate out of home showed a higher prevalence of overweight and obesity than those who ate at home (P < 0.05) , and the prevalence of overweight and obesity among them were 38.9% (1 991/5 117) and 31.7% (3 389/10 684), respectively. While women who ate at home showed a higher prevalence of overweight and obesity than those who ate out of home, and the prevalence of overweight and obesity among them were 38.1% (5 174/13 571) and 35.6% (1 581/4 445), respectively. Compared with at-home eating group, 130.4 kJ energy, 12.2 g fat, 6.2 g protein and 67.1 mg sodium were excessively consumed per day for men, and 102.5 kJ energy, 8.6 g fat, 3.4 g protein and 60.6 mg sodium were excessively consumed per day for women. To sum up, OH eating was positively associated with overweight and obesity among men (OR = 1.18, 95% CI:1.09-1.27) , but not among women (OR = 0.94, 95% CI:0.87-1.01). CONCLUSION: OH eating was positively associated with overweight and obesity among men in China.