As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.
Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; metabolism ; Proteomics ; Signal Transduction ; Ubiquitin ; metabolism ; Ubiquitin-Specific Proteases ; metabolism

Proteolysis targeting chimera (PROTAC) refers to heterobifunctional small molecules that can simultaneously bind an E3 ubiquitin ligase and a target protein, enabling specific degradation of the target protein with the aid of the ubiquitin proteasome system. At present, most PROTAC drugs are in the clinical trial stage, and the ligands are mainly non-covalent compounds. PROTAC drugs have the advantage of overcoming drug resistance and degrading "undruggable" target proteins, but non-covalent ligands could lead to the hook effect that undermines drug efficacy. With its own advantages, covalent ligands can avoid the occurrence of this phenomenon, which is of great help to the development of PROTAC. This review summarizes the progress in preclinical and clinical research and application of PROTAC molecules targeting three different classes of protein targets, including intranuclear, transmembrane, and cytosolic proteins. We also offer perspective discussions to provide research ideas and references for the future development of PROTAC.
Proteolysis ; Proteolysis Targeting Chimera ; Proteasome Endopeptidase Complex/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Proteins/metabolism* ; Ligands

Ubiquitin-proteasome system (UPS) plays an essential role in eukaryotic protein cycle,the dysregulation of which can lead to tumorigenesis.Increased activities of UPS have been observed in the patients with cancers including leukemia.UPS inhibitors can kill cancer cells by affecting ubiquitin-ligating enzyme E3,deubiquitinase,and protein degradation active sites of UPS.Therefore,UPS inhibitors have emerged as an important therapy for treating hematological malignancies,while they are rarely applied in the treatment of acute myeloid leukemia.This paper summarizes the research progress in the inhibitors affecting the protein ubiquitination at different stages of acute myeloid leukemia,aiming to provide new clues for the clinical treatment of acute myeloid leukemia.
Humans ; Proteasome Inhibitors/therapeutic use* ; Ubiquitin/metabolism* ; Proteasome Endopeptidase Complex/metabolism* ; Ubiquitination ; Leukemia, Myeloid, Acute/drug therapy*

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules by utilizing the ubiquitin proteasome system (UPS) to degrade proteins of interest. PROTACs have exhibited unprecedented efficacy and specificity in degrading various oncogenic proteins because of their unique mechanism of action, ability to target "undruggable" and mutant proteins. A series of PROTACs have been developed to degrade multiple key protein targets for the treatment of hematologic malignancy. Notably, PROTACs that target BCL-XL, IRAK4, STAT3 and BTK have entered clinical trials. The known PROTACs that have the potential to be used to treat various hematological malignancies are systematically summarized in this review.
Humans ; Hematologic Neoplasms/drug therapy* ; Proteasome Endopeptidase Complex/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Proteolysis Targeting Chimera

Targeted protein degradation (TPD) technology facilitates specific and efficient degradation of disease-related proteins through hijacking the two major protein degradation systems in mammalian cells: ubiquitin-proteasome system and lysosome pathway. Compared with traditional small molecule-inhibitors, TPD-based drugs exhibit the characteristics of a broader target spectrum. Compared with techniques interfere with protein expression on the gene and mRNA level, TPD-based drugs are target-specific, efficaciously rapid, and not constrained by post-translational modification of proteins. In the past 20 years, various TPD-based technologies have been developed. Most excitingly, two TPD-based therapeutic drugs have been approved by FDA for phase Ⅰ clinical trials in 2019. Despite of the early stage characteristics and various obstructions of the TPD technology, it could serve as a powerful tool for the development of novel drugs. This review summarizes the advances of different degradation systems based on TPD technologies and their applications in disease therapy. Moreover, the advantages and challenges of various technologies were discussed systematically, with the aim to provide theoretical guidance for further application of TPD technologies in scientific research and drug development.
Animals ; Proteasome Endopeptidase Complex/metabolism* ; Protein Processing, Post-Translational ; Proteins/metabolism* ; Proteolysis ; Technology

This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein β1, β1i, β2, β2i, β5, β5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of β1i,β2, β5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.
Apoptosis ; drug effects ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Humans ; Multiple Myeloma ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; pharmacology

Humans ; Oxidative Stress ; genetics ; Proteasome Endopeptidase Complex ; metabolism ; Protein Modification, Translational ; genetics ; Ubiquitination ; genetics

OBJECTIVETo investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.

METHODSPrimers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.

RESULTSThe enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.

CONCLUSIONNeither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.

Blotting, Western ; Cell Line ; Humans ; Mitochondria ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; SUMO-1 Protein ; metabolism ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism

Bacterial Proteins ; metabolism ; Inflammation ; metabolism ; Klebsiella pneumoniae ; metabolism ; MAP Kinase Signaling System ; Proteasome Endopeptidase Complex ; metabolism ; Ubiquitin ; metabolism

OBJECTIVETo investigate the effect of noninvasive positive pressure ventilation( NIPPy) on the gene and protein expression of biquitin-proteasome of skeletal muscle in patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD).

METHODSSeven patients with AECOPD by NIPPV were used as the study group, meanwhile, 6 patients with AECOPD who refused NIPPV was the control group. The blood gas analysis, heart rate (HR) and mean arterial pressure (MBp) were monitored before and 14 days after treatment. A skeletal muscle biopsy was performed after 14 days of therapy. The mRNA expression of ribosomal protein S21 (RPS21), Ubiquitin, Ubiquitin combined with enzyme E2 (E2), Ubiquitin ligase E3 (E3) in skeletal muscle cell were measured by RT-PCR. The protein expression of mitochondrial aconitase (AC02), protease C3 (C3), ribosomal protein SLC16 (SLC16) were detected by Western blot.

RESULTSForteen days after treatment, the patients in NIPPV group got much better improvement in PaCO2, PaO2 and HR than that of the patients.in the control group (P < 0.05). The gene expression of RPS21,Ubiquitin, E2 and E3 in skeletal muscle cell on patients with NIPPV were obviously lower than that of the control group (P < 0.05, P < 0.01). Compared with that of the control group, the protein expression of C3 and AC2 increased significantly in the NIPPV group (P < 0.01). The protein expression of SLC16 was significantly lowered in the treated group (P < 0.01).

CONCLUSIONNIPPV can ameliorate the proteasome pathway and energy metabolic disorders in patients with AECOPD.

Humans ; Muscle, Skeletal ; metabolism ; Positive-Pressure Respiration ; Proteasome Endopeptidase Complex ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; therapy ; Ubiquitin ; metabolism