Inhibition of the immunoproteasome LMP_(2) ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

The dichotomous role of immunoproteasome in cancer:Friend or foe?

Immunoproteasome is a variant of proteasome with structural differences in 20S subunits optimizing them for the production of antigenic peptides with higher binding affinity to major histocompatibility complex(MHC)-I molecules.Apart from this primary function in antigen presentation,immunoproteasome is also responsible for the degradation of proteins,both unfolded proteins for the maintenance of protein homeostasis and tumor suppressor proteins contributing to tumor progression.The altered expression of immunoproteasome is frequently observed in cancers;however,its expression levels and effects vary among different cancer types exhibiting antagonistic roles in tumor development.This review focuses on the dichotomous role of immunoproteasome in different cancer types,as well as summarizes the current progression in immunoproteasome activators and inhibitors.Specifically targeting immunoproteasome may be a beneficial therapeutic intervention in cancer treatment and understanding the role of immunoproteasome in cancers will provide a significant therapeutic insight for the prevention and treatment of cancers.

Mallory-Denk body pathogenesis revisited

This editorial reviews the recent evidence showing that Mallory-Denk bodies(MDBs)form in hepatocytes as the result of a drug-induced shift from the 26s proteasome formation to the immunoproteasome formation.The shift is the result of changes in gene expression induced in promoter activation,which is induced by the IFNγ and TNFa signaling pathway.This activates TLR 2 and 4 receptors.The TLR signaling pathway stimulates both the induction of a cytokine proinflammatory response and an up regulation of growth factors.The MDB-forming hepatocytes proliferate as a result of the increase in growth factor expression by the MDBforming cells,which selectively proliferate in response to drug toxicity.All of these mechanisms are induced by drug toxicity,and are prevented by feeding the methyl donors SAMe and betaine,supporting the epigenetic response of MDB formation.

The role of proteasomes in tumorigenesis

Protein homeostasis is the basis of normal life activities,and the proteasomefamily plays an extremely important function in this process.The proteasome 2os is a concentric circle structure with twoαrings and twoβrings overlapped.The proteasome 2os can perform both ATP-dependent and non-ATP-dependent ubiquitination proteasome degradation by binding to various subunits(such as 19S,11S,and 200 PA),which is performed by its active subunitβ1,β2,andβ5.The proteasome can degrade misfolded,excess proteins tomaintain homeostasis.At the same time,it can be utilized by tumors to degrade over-proliferate and unwanted proteins to support their growth.Proteasomes can affect the development of tumors from several aspects including tumor signaling pathways such as NF-kB and p53,cell cycle,immune regulation,and drug resistance.Proteasome-encoding genes have been found to be overexpressed in a variety of tumors,providing a potential novel target for cancer therapy.In addition,proteasome inhibitors such as bortezomib,carfilzomib,and ixazomib have been put into clinical application as the first-line treatment of multiple myeloma.More and more studies have shown that it also has different therapeutic effects in other tumors such as hepatocellular carcinoma,non-small cell lung cancer,glioblastoma,and neuroblastoma.However,proteasome inhibitors are not much effective due to their tolerance and singleness in other tumors.Therefore,further studies on their mechanisms of action and drug interactions are needed to investigate their therapeutic potential.

Dopaminergic neurons show increased low-molecular-mass protein 7 activity induced by 6-hydroxydopamine in vitro and in vivo

Background:Abnormal expression of major histocompatibility complex class I(MHC-I)is increased in dopaminergic(DA)neurons in the substantia nigra(SN)in Parkinson’s disease(PD).Low-molecular-mass protein 7(β5i)is a proteolytic subunit of the immunoproteasome that regulates protein degradation and the MHC pathway in immune cells.Methods:In this study,we investigated the role of β5i in DA neurons using a 6-hydroxydopamine(6-OHDA)model in vitro and vivo.Results:We showed that 6-OHDA upregulatedβ5i expression in DA neurons in a concentration-and time-dependent manner.Inhibition and downregulation ofβ5i induced the expression of glucose-regulated protein(Bip)and exacerbated 6-OHDA neurotoxicity in DA neurons.The inhibition of β5i further promoted the activation of Caspase 3-related pathways induced by 6-OHDA.β5i also activated transporter associated with antigen processing 1(TAP1)and promoted MHC-I expression on DA neurons.Conclusion:Taken together,our data suggest that β5i is activated in DA neurons under 6-OHDA treatment and may play a neuroprotective role in PD.

Proteasomal adaptations to FDA-approved proteasome inhibitors:a potential mechanism for drug resistance?

With proteasome inhibitors(PIs)becoming clinically available since 2003,outcomes for patients with multiple myeloma(MM)have dramatically changed,improving quality of life and survival.Despite the impressive treatment success,however,almost all MM patients who initially respond to these PIs eventually develop resistance.Furthermore,a portion of MM patients is inherently unresponsive to the PIs.Extensive mechanistic investigations identified several non-proteasomal signaling pathways suspected to be linked to the PI resistance,for which several excellent reviews are currently available.On the other hand,it is still unclear how cancer cells under high PI environments adapt to spare proteasome activity essential for survival and proliferation regardless of cancer evolution stages.This review outlines current progress towards understanding the proteasomal adaptations of cells in response to PI treatment to maintain necessary proteasome activity.A better understanding of cellular proteasomal changes in response to the PIs could provide a rationale to develop new therapeutics that could be used to overcome resistance to existing PI drugs.