The 26S proteasome at the center of the ubiquitin-proteasome system (UPS) is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.
Animals ; Humans ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Phosphorylation ; genetics ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Protein Kinases ; genetics ; metabolism

OBJECTIVETo investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia.

METHODSHyperthermic HepG2 cell models established by exposure of the cells to 42 degrees celsius; for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed.

RESULTSROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001).

CONCLUSIONThe intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.

HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Hot Temperature ; Humans ; Proteasome Endopeptidase Complex ; metabolism ; RNA, Small Interfering ; genetics ; Reactive Oxygen Species ; metabolism ; Up-Regulation

Viral infection induces numerous tripartite motif (TRIM) proteins to control antiviral immune signaling and viral replication. Particularly, SPRY-containing TRIM proteins are found only in vertebrates and they control target protein degradation by their RING-finger and SPRY domains, and proper cytoplasmic localization. To understand TRIM30 function, we analyzed its localization pattern and putative roles of its RING-finger and SPRY domains. We found that TRIM30 is located in actin-mediated cytoplasmic bodies and produces colocalized ubiquitin chains in SPRY domain- and RING-finger domain-dependent ways that are degraded by autophagy and the proteasome. These results suggest a TRIM protein-dependent degradation mechanism by cytoplasmic body formation with actin networks.
Amino Acid Sequence ; Animals ; Autophagy ; Cell Line ; Inclusion Bodies/*metabolism ; Intracellular Signaling Peptides and Proteins/chemistry/genetics/*metabolism ; Mice ; Molecular Sequence Data ; Polyubiquitin/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Proteolysis ; RING Finger Domains

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into beta-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.
Alzheimer Disease/drug therapy/metabolism ; Amyloid beta-Peptides/metabolism ; Amyotrophic Lateral Sclerosis/drug therapy/metabolism ; Animals ; Autophagy/drug effects ; DNA-Binding Proteins/metabolism ; Humans ; Huntington Disease/drug therapy/genetics/metabolism ; Lysosomes/metabolism ; Molecular Targeted Therapy ; Mutation ; Nerve Tissue Proteins/genetics/metabolism ; Neurodegenerative Diseases/drug therapy/*metabolism ; Parkinson Disease/drug therapy/metabolism ; PrPSc Proteins/metabolism ; Prion Diseases/drug therapy/metabolism ; Proteasome Endopeptidase Complex/metabolism ; Proteolysis ; Proteostasis Deficiencies/metabolism ; Superoxide Dismutase/metabolism ; Ubiquitin/metabolism ; alpha-Synuclein/metabolism ; tau Proteins/metabolism

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.
Carcinoma, Hepatocellular/*metabolism ; Cyclin-Dependent Kinase Inhibitor p27/genetics/*metabolism ; Cyclins/genetics/metabolism ; *Cytokinesis ; Gene Silencing ; Hep G2 Cells ; Humans ; Kinesin/genetics/*metabolism ; Liver Neoplasms/*metabolism ; Oncogene Proteins/genetics/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; RNA, Messenger/genetics/metabolism ; S-Phase Kinase-Associated Proteins/genetics/metabolism ; *Ubiquitination

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.
Free Radicals ; metabolism ; HeLa Cells ; Humans ; Oxidation-Reduction ; Oxidative Stress ; genetics ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Protein Binding ; Protein Modification, Translational ; genetics ; Ubiquitin ; metabolism ; Ubiquitination ; genetics

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

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.
Animals ; *Bandages ; Base Sequence ; DNA, Complementary/analysis ; Dogs/*genetics/metabolism ; Gene Expression Profiling/veterinary ; Gene Expression Regulation ; Histocompatibility Antigens Class II/*genetics/metabolism ; Molecular Sequence Data ; Omentum/*metabolism ; Proteasome Endopeptidase Complex/*genetics/metabolism ; RNA, Messenger/analysis ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; *Wound Healing

OBJECTIVETo predict and verify the target gene of miR-27a in pancreatic cancer by combining the result of comparative proteome analysis.

METHODSThe bioinformatics softwares of TargetScan,PicTar and miRanda were used to predict the possible target genes of miR-27a. Based on the results of comparative proteomics analysis, possible candidates of the target genes were selected. Expression vector of target gene 3'UTR was constructed, and then target gene was verified by dual-luciferase reporter assay system. The PANC-1 and BxPC-3 pancreatic cancer cells were treated with miR-27a mimics or negative control for 48 h. Western blot analysis was used to verify alterations of protein expression of the genes.

RESULTSPSMA1 was selected as the candidate target gene of miR-27a by bioinformatics prediction and comparative proteome analysis. Dual-luciferase reporter assay showed that miR-27a decreased luciferase activity in cells co-transfected with pmirGLO-PSMA1-WT, compared to the negative control, although significant difference of luciferase activity was not observed in cells co-transfected with pmirGLO-PSMA1-MUT between the two groups. The protein level of PSMA1 was down-regulated in pancreatic cancer cells transfected with miR-27a mimics in comparison with pancreatic cancer cells transfected with negative control.

CONCLUSIONPSMA1 is the direct target gene of miR-27a in pancreatic cancer.

3' Untranslated Regions ; genetics ; Cell Line, Tumor ; Down-Regulation ; Genetic Vectors ; HEK293 Cells ; Humans ; Luciferases ; metabolism ; MicroRNAs ; genetics ; Pancreatic Neoplasms ; metabolism ; pathology ; Plasmids ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo study the effect of human proteasome subunit Α7(PSMA7)gene silencing by small interfering RNA(siRNA)on human myeloid leukemia cell line K562.

METHODSPSMA7 gene-specific siRNA was chemically synthesized and transfected into K562 cell line by HiPerFect. The expression level of PSMA7 protein was detected by Western blot analysis. Cell proliferation was determined by MTS and cell counting. Cell cycle distribution was measured by flow cytometry. The expressions of Cyclin A, D, and E were detected by Western blot analysis. The apoptotic ratio was determined by flow cytometry.

RESULTSPSMA7 protein was evidently silenced 48 hours after transfection of the PSMA7-specific siRNA into K562 cell line. The proliferation of the cells was markedly inhibited, and the percentage of S phase cells decreased. However, no apoptosis was observed. The expressions of Cyclin A and E were down-regulated.

CONCLUSIONKnockdown of PSMA7 down-regulates the expression of Cyclin A and E and thus decreases the proportion of cells in S phase as a result, the proliferation of K562 cell line is inhibited.

Apoptosis ; Cell Cycle ; Cell Line ; Cell Proliferation ; genetics ; Down-Regulation ; Gene Knockdown Techniques ; Humans ; K562 Cells ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; therapeutic use ; Transfection