PGK1-coupled HSP90 stabilizes GSK3βexpression to regulate the stemness of breast cancer stem cells

Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.

Pathophysiology of obesity and its associated diseases

The occurrence of obesity has increased across the whole world. Many epidemiological studies have indicated that obesity strongly contributes to the development of cancer, cardiovascular diseases, type 2 diabetes, liver diseases and other disorders, accounting for a heavy burden on the public and on health-care systems every year. Excess energy uptake induces adipocyte hypertrophy, hyperplasia and formation of visceral fat in other non-adipose tissues to evoke cardiovascular disease, liver diseases. Adipose tissue can also secrete adipokines and inflammatory cytokines to affect the local microenvironment,induce insulin resistance, hyperglycemia, and activate associated inflammatory signaling pathways. This further exacerbates the development and progression of obesity-associated diseases. Although some progress in the treatment of obesity has been achieved in preclinical and clinical studies, the progression and pathogenesis of obesity-induced diseases are complex and unclear. We still need to understand their links to better guide the treatment of obesity and associated diseases. In this review, we review the links between obesity and other diseases, with a view to improve the future management and treatment of obesity and its co-morbidities.

In silico design of anti-tumor mini-protein targeting MDM2

We designed a disulfide-crosslinked mini-protein with a two-helical topology consisting of L-and Damino acids,which was exceptionally stable in serum.Therefore,we further used it as a scaffold to design mini-proteins targeting p53 positive tumor cells.Based on bifunctional grafting,key residues from the transactivation domain of p53 and a designed unnatural amino acid were grafted into the helix constituted by L-amino acids to confer the mini-protein with MDM2 inhibitory activity.Meanwhile,ten Arg residues were introduced to improve its membrane penetrating capacity.Among the mini-proteins,UPROL-10e showed nano-molar binding affinity on MDM2 and cellular toxicity on p53 expressing HCT116cells.

MomL inhibits bacterial antibiotic resistance through the starvation stringent response pathway

Antibiotic resistance in gram-negative pathogens has become one of the most serious global public health threats.The role of the N-acyl homoserine lactone(AHL)-mediated signaling pathway,which is widespread in gram-negative bacteria,in the bacterial resistance process should be studied in depth.Here,we report a degrading enzyme of AHLs,MomL,that inhibits the antibiotic resistance of Pseudomonas aeruginosa through a novel mechanism.The MomL-mediated reactivation of kanamycin is highly associated with the relA-mediated starvation stringent response.The degradation of AHLs by MomL results in the inability of LasR to activate relA,which,in turn,stops the activation of downstream rpoS.Further results show that rpoS directly regulates the type VI secretion system H2-T6SS.Under MomL treatment,inactivated RpoS fails to regulate H2-T6SS;therefore,the expression of effector phospholipase A is reduced,and the adaptability of bacteria to antibiotics is weakened.MomL in combination with kanamycin is effective against a wide range of gram-negative pathogenic bacteria.Therefore,this study reports a MomL-antibiotic treatment strategy on antibiotic-resistant bacteria and reveals its mechanism of action.

Molecular dynamic simulation on the conformation of mouse muscle type nAChR

A mouse muscle type nAChR model((α1)_(2)βδg)was built based on the cryoelectron microscopic structure of intact Torpedo marmorata nAChR and the high resolution crystal structure of nAChR-α1 subunit.The conformation of the pentameric nAChR model was investigated by molecular dynamic simulation.The function of water molecule in the hydrophilic interior was clarified.The reason for Tyr127 showing two alternative conformations was discussed in detail.

Mechanism of interactions betweenα-conotoxin RegIIA and carbohydrates at the humanα3β4 nicotinic acetylcholine receptor

Conotoxins are marine peptide toxins from marine cone snails.Theα-conotoxin RegIIA can selectively act on human(h)α3β4 nicotinic acetylcholine receptor(nAChR),and is an important lead for drug development.The high-resolution cryo-electron microscopy structure of theα3β4 nAChR demonstrates several carbohydrates are located near the orthosteric binding sites,which may affectα-conotoxin binding.Oligosaccharide chains can modify the physical and chemical properties of proteins by changing the conformation,hydrophobicity,quality and size of the protein.The purpose of this study is to explore the effect of oligosaccharide chains on the binding modes and activities of RegIIA and its derivatives at hα3β4 nAChRs.Through computational simulations,we designed and synthesized RegIIA mutants at position 14 to explore the importance of residue H14 to the activity of the peptide.Molecular dynamics simulations suggest that the oligosaccharide chains affect the binding of RegIIA at the hα3β4 nAChR through direct interactions with H14 and by affecting the C-loop conformation of the binding sites.Electrophysiology studies on H14 analogues suggest that in addition to forming direct interactions with the carbohydrates,the residue might play an important role in maintaining the conformation of the peptide.Overall,this study further clarifies the structure–activity relationship ofα-conotoxin RegIIA at the hα3β4 nAChR and,also provides important experimental and theoretical basis for the development of new peptide drugs.