Ubiquitin-proteasome system and oxidative stress in liver transplantation

A major issue in organ transplantation is the development of a protocol that can preserve organs under optimal conditions. Damage to organs is commonly a consequence of flow deprivation and oxygen starvation following the restoration of blood flow and reoxygenation. This is known as ischemia-reperfusion injury(IRI): a complex multifactorial process that causes cell damage. While the oxygen deprivation due to ischemia depletes cell energy, subsequent tissue oxygenation due to reperfusion induces many cascades, from reactive oxygen species production to apoptosis initiation. Autophagy has also been identified in the pathogenesis of IRI, although such alterations and their subsequent functional significance are controversial. Moreover, proteasome activation may be a relevant pathophysiological mechanism. Different strategies have been adopted to limit IRI damage, including the supplementation of commercial preservation media with pharmacological agents or additives. In this review, we focus on novel strategies related to the ubiquitin proteasome system and oxidative stress inhibition, which have been used to minimize damage in liver transplantation.

Peroxisome proliferator activated receptor-γ and the ubiquitin-proteasome system in colorectal cancer

Peroxisome proliferator activated receptor-γ (PPARγ), a transcription factor of the nuclear receptor superfamily plays a significant role in colorectal cancer pathogenesis. In most experimental systems PPARγ activation has tumor suppressing effects in the colon. PPARγ is regulated at multiple levels by the ubiquitin-proteasome system (UPS). At a first level, UPS regulates PPARγ transcription. This regulation involves both PPARγ transcription specific factors and the general transcription machinery. At a second level UPS regulates PPARγ and its co-factors themselves, as PPARγ and many co-factors are proteasome substrates. At a third level of regulation, transduction pathways working in parallel but also having interrelations with PPARγ are regulated by the UPS, creating a network of regulation in the colorectal carcinogenesisrelated pathways that are under UPS control. Activation of PPARγ transcription by direct pharmacologic activators and by stabilization of its molecule by proteasome inhibitors could be strategies to be exploited in colorectal cancer treatment.

Ubiquitin proteasome system research in gastrointestinal cancer

The ubiquitin proteasome system(UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies,including esophageal,gastric and colorectal cancers. Here,we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer,highlighting its role in tumor occurrence and development,providing information for therapeutic targets research and antigastrointestinal tumor drug design.

Administration of grape seed extract alleviates age-associated decline in ubiquitin-proteasome system and cardiomyocyte apoptosis in rats

Effective clearance of oxidized, damaged, and/or misfolded proteins in the cell by the ubiquitin-proteasome system (UPS) is critical for cell homeostasis, survival and function. We hypothesized that in the aging heart, generation of free radicals could impair UPS where the associated build-up of polyubiquitinated proteins could trigger programmed cell death. To test this, we used young (4 months old) and aged (24 months old) rats to analyze polyubiquitinated proteins, proteasome activity and programmed cell death in the ventricular tissue samples. Our studies reveal excessive deposition of polyubiquitinated proteins in the ventricular tissue extracts of old rats when compared to younger rats. The increased ubiquitination was accompanied by a significant decrease in 20S proteasome activity. Since the loss of proteasome-mediated clearance of ubiquitinated proteins is linked to programmed cell death, we measured TUNEL activity in aged rat heart and compared with younger animals. Aged animal hearts showed a substantial increase in programmed cell death as evidenced by TUNEL positive nuclei and DNA fragmentation. Analyses of cell death/survival pathways support our findings in terms of age-associated increase in the nuclear localization of p53, Bax/Bcl2 ratio and cleaved (active) caspase-3 and decreased expression of cellular inhibitor of apoptosis (cIAP1). Administration of grape seed extract (GSE) as a source of antioxidants significantly reduced these age-associated deleterious changes suggesting that free radicals primarily contribute to impaired UPS function and increased programmed cell death and that administration of antioxidants during aging could protect cardiac muscle cells and preserve ventricular function.

Neuroendocrine differentiation and the ubiquitinproteasome system in cancer:Partners or enemies?

Neuroendocrine(NE)differentiation of cancer and deregulation of the ubiquitin-proteasome system(UPS)are two processes that have been independently linked to the development of aggressive and treatment-resistant tumors.Striking data suggest a plausible interconnection between these two mechanisms,based on indirect evidence of neuropeptide-induced effects on UPS,reversed by proteasome inhibition and deubiquitinaselike properties of NE markers.Deciphering the model of their exact interactions is one of the keys to targeting the NE malignant phenotype more effectively.

Emerging Role of the Ubiquitin Proteasome System in the Control of Shoot Apical Meristem Function

The shoot apical meristem （SAM） is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant＇s life. A plethora of different families of transcription factors （TFs） play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system （UPS）. However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.

The Ubiquitin-Proteasome System and Its Role in Inflammatory and Autoimmune Diseases

Protein degradation through the ubiquitin-proteasome system is the major pathway of non-lysosomal proteolysis of intracellular proteins. It plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, division, development and differentiation, apoptosis, cell trafficking, and modulation of the immune and inflammatory responses. The central element of this system is the covalent linkage of ubiquitin to targeted proteins, which are then recognized by the 26S proteasome, an adenosine triphosphate-dependent, multi-catalytic protease. Damaged, oxidized, or misfolded proteins as well as regulatory proteins that control many critical cellular functions are among the targets of this degradation process. Aberration of this system leads to the dysregulation of cellular homeostasis and the development of multiple diseases. In this review, we described the basic biochemistry and molecular biology of the ubiquitin-proteasome system, and its complex role in the development of inflammatory and autoimmune diseases. In addition, therapies and potential therapeutic targets related to the ubiquitin-proteasome system are discussed as well.

Overexpression of proteasome 26S subunit non-ATPase 6 protein and its clinicopathological significance in intrahepatic cholangiocarcinoma

BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AIM To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.METHODS The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology.Forty-two paired specimens of ICC and adjacent noncancerous tissues were collected.PSMD6 protein expression was determined by immunohistochemistry.Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level,and its association with ICC patients’various clinicopathological characteristics was investigated.RESULTS The PSMD6 gene was found to be essential for the growth of ICC cell lines.PSMD6 protein was significantly overexpressed in ICC tissues(P<0.001),but showed no significant association with patient age,gender,pathological grade,or tumor-node-metastasis stage(P>0.05).CONCLUSION PSMD6 can promote the growth of ICC cells,thus playing a pro-oncogenic role.

Is 26S proteasome non-ATPase regulatory subunit 6 a potential molecular target for intrahepatic cholangiocarcinoma?

In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents.Thus,there has been a sustained search for novel molecular targets for iCCA.The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6(PSMD6),a 19S regulatory subunit of the proteasome,in human iCCA cells and specimens.The authors employed clustered regularly interspaced short palindromic repeat(CRISPR)knockout screening technology integrated with the computational CERES algorithm,and analyzed the human protein atlas(THPA)database and tissue microarrays.The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines,and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters.The authors conclude that PSMD6 may play a promoting role in iCCA.The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening,in vivo experiments,and a discussion of plausible mechanistic cues,which,therefore,dampen the significance of the results.Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA.In addition,the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA,development of proteasome inhibitors for cancer therapy,and advantages of CRISPR screening technology,computational methods,and THPA database as experimental tools for fighting cancer.We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.

老年白内障患者晶状体中20s Proteasome的表达

目的探讨老年性白内障患者晶状体中20s Proteasome表达及酶活性的变化及其与白内障发病的关系。方法分别应用Western印迹法及荧光标记蛋白质底物法检测老年性白内障患者晶状体中20s Proteasome的表达及酶活性变化。结果随着晶状体混浊程度及核硬度的增加,20s Proteasome的表达及酶活性均呈下降趋势。结论在老年性白内障发病过程中,20s Proteasome与晶状体混浊程度相关。

Cloning and Sequence Analysis of Proteasome β5 Fragment in Helicoverpa armigera

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm (Helicoverpa armigera), this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends (RACE) technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF (843 bp) and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point(pI) of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene (GenBank accession number: FJ358434).

Evolution of COP9 Signalosome and Proteasome Lid Complex

The COP9 signalosome and the regulatory lid of the 26S proteasome are both eight-subunit protein complexes which are present in most eukaryotes. There is a one-to-one relationship between the corresponding subunits of the two protein complexes in terms of their size and amino acid sequences. Eight groups of subunits from the COP9 signalosome and the proteasome lid complex of different organisms are collected from all the databases at the NCBI website. The corresponding subunits of COP9 signalosome and proteasome lid complex share at least 12% amino acid identity and some conserved regions, and the conserved sites spread evenly over the entire length of the subunits, suggesting that the two complexes have a common evolutionary ancestor. Phylogenetic analyses based on the amino acid sequences of the corresponding subunits of two protein complexes indicate that every tree consists of two clades. The subunits from one of the two protein complexes of different organisms are grouped into one of the two clades respectively. The sequences of single-cell organisms are always the basal groups to that of multi-cell animal and plant species. These results imply that the duplication/divergence events of COP9 signalosome and regulatory lid of the proteasome genes have occurred before the divergence of single-cell and multi-cell eukaryotes, and the genes of the two complexes are independently evolved. The analyses of dN/dS correlation show significant Pearson's correlations between 21 and 15 pairs of subunit-encoding sequences within the COP9 signalosome and the proteasome lid complex respectively, suggesting that those subunits pairs might have related functions and interacted with one another, and resulted in co-evolution.

The roles of the proteasome pathway in signal transduction and neurodegenerative diseases

There are two degradation systems in mammalian cells, autophagy/lysosomal pathway and ubiquitin-proteasome pathway. Proteasome is consist of multiple protein subunits and plays important roles in degradation of short-lived cellular proteins. Recent studies reveal that proteasomal degradation system is also involved in signal transduction and regulation of various cellular functions. Dysfunction or dysregulation of proteasomal function may thus be an important pathogenic mechanism in certain neurological disorders. This paper reviews the biological functions of proteasome in signal transduction and its potential roles in neurodegenerative diseases.

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase(IKK)

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy

Proteasome inhibitor treatment in alcoholic liver disease

Oxidative stress, generated by chronic ethanol consumption, is a major cause of hepatotoxicity and liver injury. Increased production of oxygen-derived free radicals due to ethanol metabolism by CYP2E1 is principally located in the cytoplasm and in the mitochondria, which does not only injure liver cells, but also other vital organs, such as the heart and the brain. Therefore, there is a need for better treatment to enhance the antioxidant response elements. To date, there is no established treatment to attenuate high levels of oxidative stress in the liver of alcoholic patients. To block this oxidative stress, proteasome inhibitor treatment has been found to significantly enhance the antioxidant response elements of hepatocytes exposed to ethanol. Recent studies have shown in an experimental model of alcoholic liver disease that proteasome inhibitor treatment at low dose has cytoprotective effects against ethanol-induced oxidative stress and liver steatosis. The beneficial effects of proteasome inhibitor treatment against oxidative stress occurred because antioxidant response elements （glutathione peroxidase 2, superoxide dismutase 2, glutathione synthetase, glutathione reductase, and GCLC） were upregulated when rats fed alcohol were treated with a low dose of PS-34Z （Bortezomib, Velcade）. This is an important finding because proteasome inhibitor treatment up-regulated reactive oxygen species removal and glutathione recycling enzymes, while ethanol feeding alone down-regulated these antioxidant elements. For the first time, it was shown that proteasome inhibition by a highly specific and reversible inhibitor is different from the chronic ethanol feeding-induced proteasome inhibition. As previously shown by our group, chronic ethanol feeding causes a complex dysfunction in the ubiquitin proteasome pathway, which affects the proteasome system, as well as the ubiquitination system. The beneficial effects of proteasome inhibitor treatment in alcoholic liver disease are related to proteasome inhibitor reversibility and the rebound of proteasome activity 72 h post PS-341 administration.

Therapeutic proteasome inhibition in experimental acute pancreatitis

AIM： To establish the therapeutic potential of proteasome inhibition, we examined the therapeutic effects of MG132 （Z-Leu-Leu-Leu-aldehyde） in an experimental model of acute pancreatitis. METHODS： Pancreatitis was induced in rats by two hourly intraperitoneal （ip） injections of cholecystokinin octapeptide （CCK; 2×100μg/kg） and the proteasome inhibitor MG132 （10 mg/kg ip） was administered 30 min after the second CCK injection. Animals were sacrificed 4 h after the first injection of CCK. RESULTS： Administering the proteasome inhibitor MG132 （at a dose of 10 mg/kg, ip） 90 min after the onset of pancreatic inflammation induced the expression of cell-protective 72 kDa heat shock protein （HSP72） and decreased DNA-binding of nuclear factor-κB （NF-κB）. Furthermore MG132 treatment resulted in milder inflammatory response and cellular damage, as revealed by improved laboratory and histological parameters of pancreatitis and associated oxidative stress. CONCLUSION： Our findings suggest that proteasome inhibition might be beneficial not only for the prevention, but also for the therapy of acute pancreatitis.

Epigenetics of proteasome inhibition in the liver of rats fed ethanol chronically

AIM： TO examine the effects of ethanol-induced proteasome inhibition, and the effects of proteasome inhibition in the regulation of epigenetic mechanisms.METHODS： Rats were fed ethanol for 1 mo using the Tsukamoto-French model and were compared to rats given the proteasome inhibitor PS-341 （Bortezomib, Velcade^TM） by intraperitoneal injection. Microarray analysis and real time PCR were performed and proteasome activity assays and Western blot analysis were performed using isolated nuclei.RESULTS： Chronic ethanol feeding caused a significant inhibition of the ubiquitin proteasome pathway in the nucleus, which led to changes in the turnover of transcriptional factors, histone-modifying enzymes, and, therefore, affected epigenetic mechanisms. Chronic ethanol feeding was related to an increase in histone acetylation, and it is hypothesized that the proteasome proteolytic activity regulated histone modifications by controlling the stability of histone modifying enzymes, and, therefore, regulated the chromatin structure, allowing easy access to chromatin by RNA polymerase, and, thus, proper gene expression. Proteasome inhibition by PS-341 increased histone acetylation similar to chronic ethanol feeding. In addition, proteasome inhibition caused dramatic changes in hepatic remethylation reactions as there was a significant decrease in the enzymes responsible for the regeneration of S-adenosylmethionine, and, in particular, a significant decrease in the betaine-homocysteine methyltransferase enzyme. This suggested that hypomethylation was associated with proteasome inhibition, as indicated by the decrease in histone methylation.CONCLUSION： The role of proteasome inhibition in regulating epigenetic mechanisms, and its link to liver injury in alcoholic liver disease, is thus a promising approach to study liver injury due to chronic ethanol consumption.

Physiological levels of ATP negatively regulate proteasome function

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is -100 μM. IntraceUular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration （between 50 and 100μM） stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intraceHular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.

Implication of altered proteasome function in alcoholic liverinjury

The proteasome is a major protein-degrading enzyme, which catalyzes degradation of oxidized and aged proteins, signal transduction factors and cleaves peptides for antigen presentation. Proteasome exists in the equilibrium of 26S and 20S particles. Proteasome function is altered by ethanol metabolism, depending on oxidative stress levels： low oxidative stress induces proteasome activity, while high oxidative stress reduces it. The proposed mechanisms for modulation of proteasome activity are related to oxidative modification of proteasomal proteins with primary and secondary products derived from ethanol oxidation. Decreased proteolysis by the proteasome results in the accumulation of insoluble protein aggregates, which cannot be degraded by proteasome and which further inhibit proteasome function. Mallory bodies, a common signature of alcoholic liver diseases, are formed by liver cells, when proteasome is unable to remove cytokeratins. Proteasome inhibition by ethanol also promotes the accumulation of pro-apoptotic factors in mitochondria of ethanol-metabolizing liver cells that are normally degraded by proteasome. In addition, decreased proteasome function also induces accumulation of the negative regulators of cytokine signaling （I-~B and SOCS）, thereby blocking cytokine signal transduction. Finally, ethanol-elicited blockade of interferon type 2 and 2 signaling and decreased proteasome function impairs generation of peptides for MHC class Ⅰ-restricted antigen presentation.

Effects of ethanol on the proteasome interacting proteins

Proteasome dysfunction has been repeatedly reported in alcoholic liver disease. Ethanol metabolism endproducts affect the structure of the proteasome, and, therefore, change the proteasome interaction with its regulatory complexes 19S and PA28, as well as its interacting proteins. Chronic ethanol feeding alters the ubiquitin-proteasome activity by altering the interaction between the 19S and the 20S proteasome interaction. The degradation of oxidized and damaged proteins is thus decreased and leads to accumulation of insoluble protein aggregates, such as Mallory-Denk bodies. Ethanol also affects the immunoproteasome formation. PA28a/b interactions with the 20S proteasome are decreased in the proteasome fraction isolated from the liver of rats fed ethanol chronically, thus affecting the cellular antigen presentation and defense against pathogenic agents. Recently, it has been shown that ethanol also affects the proteasome interacting proteins (PIPs). Interaction of the proteasome with Ecm29 and with deubiquitinating enzymes Rpn11, UCH37, and Usp14 has been found to decrease. However, the two UBL-ubiquitin-associated domain (UBA) PIPs p62 and valosin-containing protein are upregulated when the proteasome is inhibited. The increase of these UBL-UBA proteins, as well as the increase in Hsp70 and Hsp25 levels, compensated for the proteasome failure and helped in the unfolding/docking of misfolded proteins. Chronic alcohol feeding to rats causes a significant inhibition of the proteasome pathway and this inhibition results from a decreases of the interaction between the 20S proteasome and the regulatory complexes, PIPs, and the ubiquitin system components.