Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic prot...Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint (PMF) data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.展开更多
Multiple myeloma(MM) is a cancer caused by uncontrolled proliferation of antibody-secreting plasma cells in bone marrow, which represents the second most common hematological malignancy. MM is a highly heterogeneous d...Multiple myeloma(MM) is a cancer caused by uncontrolled proliferation of antibody-secreting plasma cells in bone marrow, which represents the second most common hematological malignancy. MM is a highly heterogeneous disease and can be classified into a spectrum of subgroups based on their molecular and cytogenetic abnormalities. In the past decade, novel therapies, especially, the first-in-class proteasome inhibitor bortezomib, have been revolutionary for the treatment of MM patients. Despite these remarkable achievements, myeloma remains incurable with a high frequency of patients suffering from a relapse, due to drug resistance. Mutation in the proteasome β5-subunit(PSMB5) was found in a bortezomib-resistant cell line generated via long-term coculture with increasing concentrations of bortezomib in 2008, but their actual implication in drug resistance in the clinic has not been reported until recently. A recent study discovered four resistance-inducing PSMB5 mutations from a relapsed MM patient receiving prolonged bortezomib treatment. Analysis of the dynamic clonal evolution revealed that two subclones existed at the onset of disease, while the other two subclones were induced. Protein structural modeling and functional assays demonstrated that all four mutations impaired the binding of bortezomib to the 20 S proteasome, conferring different degrees of resistance. The authors further demonstrated two potential approaches to overcome drug resistance by using combination therapy for targeting proteolysis machinery independent of the 20 S proteasome.展开更多
OBJECTIVE To investigate the effects of T-006(tetramethylpyrazine derivative)in promotingα-Synuclein(α-Syn)degradation and evaluated the neuroprotective effects in cellular and animalα-Syn model of Parkinson diseas...OBJECTIVE To investigate the effects of T-006(tetramethylpyrazine derivative)in promotingα-Synuclein(α-Syn)degradation and evaluated the neuroprotective effects in cellular and animalα-Syn model of Parkinson disease(PD).METHODS The inducible PC12 cells overexpressingα-syn and the homozygous transgenic(Tg)mice expressing A53T humanα-syn were used to evaluate the neuroprotective effects of T-006.For cellular study,MTT,Western blotting,proteasomal activity assay and qRT-PCR were applied to analyze the pharmacological effects and underlying mecha⁃nisms.The gene knock-down and overexpression approaches were used to dissect the molecular signaling pathways.For animal study,ten-month-old homozygousα-Syn Tg mice were treated with T-006(3 mg·kg-1)daily by gavage for four weeks.The Western blotting,immunohistochemistry and behavioral tests were applied to determine the neuropatho⁃logical changes.RESULTS T-006 promoted the degradation of WT and mutantα-Syn in PC12α-Syn inducible cells via an ubiquitin-proteasome system(UPS)dependent and autophagy-lysosome pathway independent manner.The mecha⁃nism of action involved the upregulation of 20S proteasome subunit LMP7 expression,which leads to activation of the chymotrypsin-like proteasomal activity for protein degradation.Mechanistically,we demonstrated that T-006 activated PKA/Akt/mTOR pathway upstream for LMP 7 up-regulation and UPS activation.Finally,we illustrated that T-006 promoted both Triton-soluble and-insoluble forms ofα-syn and protected againstα-Syn-induced neurotoxicity in A53Tα-Syn Tg mice.CONCLUSION T-006 is a potent UPS activator which promotes the degradation of pathogenic proteinα-Syn in cellular and animal PD models.Our study thus high-lights the therapeutic potential of small molecular UPS activator like T-006 in the treatment of PD and related conditions.展开更多
基金the Science and Technology Commission Foundation of Jilin Province,No.200505200the Distinguished Professor Foundation of Jilin University,No.450011011204
文摘Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint (PMF) data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.
文摘Multiple myeloma(MM) is a cancer caused by uncontrolled proliferation of antibody-secreting plasma cells in bone marrow, which represents the second most common hematological malignancy. MM is a highly heterogeneous disease and can be classified into a spectrum of subgroups based on their molecular and cytogenetic abnormalities. In the past decade, novel therapies, especially, the first-in-class proteasome inhibitor bortezomib, have been revolutionary for the treatment of MM patients. Despite these remarkable achievements, myeloma remains incurable with a high frequency of patients suffering from a relapse, due to drug resistance. Mutation in the proteasome β5-subunit(PSMB5) was found in a bortezomib-resistant cell line generated via long-term coculture with increasing concentrations of bortezomib in 2008, but their actual implication in drug resistance in the clinic has not been reported until recently. A recent study discovered four resistance-inducing PSMB5 mutations from a relapsed MM patient receiving prolonged bortezomib treatment. Analysis of the dynamic clonal evolution revealed that two subclones existed at the onset of disease, while the other two subclones were induced. Protein structural modeling and functional assays demonstrated that all four mutations impaired the binding of bortezomib to the 20 S proteasome, conferring different degrees of resistance. The authors further demonstrated two potential approaches to overcome drug resistance by using combination therapy for targeting proteolysis machinery independent of the 20 S proteasome.
基金Science and Technology Development Fund(FDCT)of Macao SAR(069/2015/A2134/2014/A3+4 种基金062-2017-AIR)Research Committee,University of Macao(MYRG2015-00182-ICMS-QRCMMYRG2015-00214-ICMSQRCMMYRG139(Y1-L4)-ICMS12-LMYand MYRG2016-00129-ICMS-QRCM)
文摘OBJECTIVE To investigate the effects of T-006(tetramethylpyrazine derivative)in promotingα-Synuclein(α-Syn)degradation and evaluated the neuroprotective effects in cellular and animalα-Syn model of Parkinson disease(PD).METHODS The inducible PC12 cells overexpressingα-syn and the homozygous transgenic(Tg)mice expressing A53T humanα-syn were used to evaluate the neuroprotective effects of T-006.For cellular study,MTT,Western blotting,proteasomal activity assay and qRT-PCR were applied to analyze the pharmacological effects and underlying mecha⁃nisms.The gene knock-down and overexpression approaches were used to dissect the molecular signaling pathways.For animal study,ten-month-old homozygousα-Syn Tg mice were treated with T-006(3 mg·kg-1)daily by gavage for four weeks.The Western blotting,immunohistochemistry and behavioral tests were applied to determine the neuropatho⁃logical changes.RESULTS T-006 promoted the degradation of WT and mutantα-Syn in PC12α-Syn inducible cells via an ubiquitin-proteasome system(UPS)dependent and autophagy-lysosome pathway independent manner.The mecha⁃nism of action involved the upregulation of 20S proteasome subunit LMP7 expression,which leads to activation of the chymotrypsin-like proteasomal activity for protein degradation.Mechanistically,we demonstrated that T-006 activated PKA/Akt/mTOR pathway upstream for LMP 7 up-regulation and UPS activation.Finally,we illustrated that T-006 promoted both Triton-soluble and-insoluble forms ofα-syn and protected againstα-Syn-induced neurotoxicity in A53Tα-Syn Tg mice.CONCLUSION T-006 is a potent UPS activator which promotes the degradation of pathogenic proteinα-Syn in cellular and animal PD models.Our study thus high-lights the therapeutic potential of small molecular UPS activator like T-006 in the treatment of PD and related conditions.
