Alteration of 20S proteasome-subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of diabetes mellitus

Insulin-dependent diabetes mellitus is known to go along with enhanced muscle protein breakdown. Since evidence has been presented that the ubiquitin-proteasome system is significantly involved in muscle wasting under this condition, we have investigated, whether this biological role goes along with alterations of the proteasome system in skeletal muscle of streptozotocin-diabetic rats. Previously, we have found a drop of overall proteasome activity in muscle extracts of rats after induction of diabetes but no change in total amount of 20S proteasome was detected. In the present investigation under the same diabetic conditions we have measured a significant decrease in the amount of proteasome activator PA28, a finding that explains the loss of total proteasome activity. Since increased mRNA levels of proteasome subunits have been measured in muscle tissue of rats after induction of diabetes, we have isolated and purified 20S proteasomes from muscle tissue of control and 6 days diabetic rats. The specific chymotrypsin-like, trypsin-like, and peptidylglutamylpeptide-hydrolysing activities of proteasomes from diabetic and control rats were found to be not significantly different. Therefore, we have fractionated 20S proteasomes into their subtypes and detected that induction of diabetes mellitus effects a redistribution of subtypes of all three proteasome populations but only the increase in subtype V (immuno-subtype) was statistically significant. This altered subtype pattern obviously meets the requirements to the system under wasting conditions. Since this process goes along with de novo biogenesis of 20S proteasomes, it most likely explains the phenomenon of elevated mRNA concentrations of proteasome subunits after induction of diabetes mellitus.     

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.     

Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis

Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.     

The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle

Proteasome activity is fine-tuned by associating the proteolytic core particle (CP) with stimulatory and inhibitory complexes. Although several mammalian regulatory complexes are known, knowledge of yeast proteasome regulators is limited to the 19-subunit regulatory particle (RP), which confers ubiquitin-dependence on proteasomes. Here we describe an alternative proteasome activator from Saccharomyces cerevisiae, Blm10. Synthetic interactions between blm10Delta and other mutations that impair proteasome function show that Blm10 functions together with proteasomes in vivo. This large, internally repetitive protein is found predominantly within hybrid Blm10-CP-RP complexes, representing a distinct pool of mature proteasomes. EM studies show that Blm10 has a highly elongated, curved structure. The near-circular profile of Blm10 adapts it to the end of the CP cylinder, where it is properly positioned to activate the CP by opening the axial channel into its proteolytic chamber.     

Evidences against vesicle-dependent trafficking and involvement of extracellular proteasomes into cell-to-cell communications

The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids of both healthy humans and patients suffering from a variety of inflammatory, autoimmune, and neoplastic diseases. The concentration of these extracellular proteasomes has been found to correlate with the diseased state, being of a prognostic significance. The transport mechanisms and functions of these proteasomes, however, are largely unclear. Previous studies revealed that the transport of extracellular proteasomes may occur via microvesicles and exosomes, which led to the hypothesis that extracellular proteasomes are implicated in cell-to-cell communication process. Here we show that microvesicles and exosomes, two major known types of intercellular vehicles, contain no detectable proteasomes. Moreover, neither affinity purified nor naturally released into conditioned medium by donor cells 20S proteasomes could penetrate recipient HeLa cells. Taken together, these results suggest that extracellular proteasomes are unlikely to be involved in the cell-to-cell communication and that their release by cells serve other biological purposes.     

Proteasome activity in tumors of the female reproductive system

Although proteasomes (multiproteinase protein complexes) are known to play an important role in cancer pathogenesis, there is little information about their activity in human tumor tissues. The chymotrypsin-like activity of proteasomes in breast cancer (BC) and endometrial cancer (EC) tissues was studied. It was shown that the chymotrypsin-like total proteasome activity and the 20S and 26S proteasome pool activities were significantly higher in malignant than in normal tissues. An increase in the size of either BC or EC tumors did not affect the proteasome activity, whereas the propagation of a malignant process did. If compared with BC non-metastatic tumors, a reliable decrease in the total activity and the 26S proteasome activity was observed in BC tumors with regional lymph node metastases. In EC tissues, the total proteasome activity and the 20S and 26S proteasome pool activities increased when the depth of tumor myometrial invasion grew. These data demonstrated that the proteasome activity significantly varied in the process of carcinogenesis. Further proteasome studies could serve as the basis for the development of new criteria for prognosis of female reproductive system cancer and the search for effective antitumor agents in targeted therapy.     

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.     

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.     

A Highlights from MBoC Selection: Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome deficiencies; when its expression is repressed, proteasome deficiencies intensify. Arp2/3 is best known for regulating membrane dynamics and vesicular transport; thus, we performed photobleaching experiments and showed that proteasomes are readily imported into the nucleus but exit the nucleus slowly. Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access substrates throughout the cell, allowing them to effectively regulate cell-compartment–specific activities.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.     

Molecular biology of proteasomes

Eukaryotic proteasomes are unusually large proteins with a heterogeneous subunit composition and have been classified into two isoforms with apparently distinct sedimentation coefficients of 20S and 26S. The 20S proteasome is composed of a set of small subunits with molecular masses of 21–32 kDa. The 26S proteasome is a multi-molecular assembly, consisting of a central 20S proteasome and two terminal subsets of multiple subunits of 28–112 kDa attached to the central part in opposite orientations. The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been deduced from the nucleotide sequences of cDNAs or genes isolated by recombinant DNA techniques. These genes constitute a unique multi-gene family encoding homologous polypeptides that have been conserved during evolution. In contrast, little is yet known about the terminal structures of the 26S proteasome, but the cDNA clonings of those of humans are currently in progress. In this review, I summarize available information of the structural features on eukaryotic 20S and 26S proteasomes which has been clarified by molecular-biological methods.     

Rethinking Proteasome Evolution: Two Novel Bacterial Proteasomes

The proteasome is a multisubunit structure that degrades proteins. Protein degradation is an essential component of regulation because proteins can become misfolded, damaged, or unnecessary. Proteasomes and their homologues vary greatly in complexity: from HslV (heat shock locus v), which is encoded by 1 gene in bacteria, to the eukaryotic 20S proteasome, which is encoded by more than 14 genes. Despite this variation in complexity, all the proteasomes are composed of homologous subunits. We searched 238 complete bacterial genomes for structures related to the proteasome and found evidence of two novel groups of bacterial proteasomes. The first, which we name Anbu, is sparsely distributed among cyanobacteria and proteobacteria. We hypothesize that Anbu must be very ancient because of its distribution within the cyanobacteria, and that it has been lost in many more recent species. We also present evidence for a fourth type of bacterial proteasome found in a few beta-proteobacteria, which we call beta-proteobacteria proteasome homologue (BPH). Sequence and structural analyses show that Anbu and BPH are both distinct from known bacterial proteasomes but have homologous structures. Anbu is encoded by one gene, so we postulate a duplication of Anbu created the 20S proteasome. Anbu's function appears to be related to transglutaminase activity, not the general stress response associated with HslV. We have found different combinations of Anbu, BPH, and HslV within these bacterial genomes, which raises questions about specialized protein degradation systems.     

Therapeutic targeting of cancer cell cycle using proteasome inhibitors

Proteasomes are multicatalytic protease complexes in the cell, involved in the non-lysosomal recycling of intra-cellular proteins. Proteasomes play a critical role in regulation of cell division in both normal as well as cancer cells. In cancer cells this homeostatic function is deregulated leading to the hyperactivation of the proteasomes. Proteasome inhibitors (PIs) are a class of compounds, which either reversibly or irreversibly block the activity of proteasomes and induce cancer cell death. Interference of PIs with the ubiquitin proteasome pathway (UPP) involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI induced cell cycle arrest in a variety of cancer cells. Although many PIs target the proteasomes, not all of them are effective in cancer therapy. Some cancers develop resistance against proteasome inhibition by possibly activating compensatory signaling pathways. However, the details of the activation of these pathways and their contribution to resistance to PI therapy remain obscure. Delineation of these pathways may help in checking resistance against PIs and deducing effective combinational approaches for improved treatment strategies. This review will discuss some of the signaling pathways related to proteasome inhibition and cell division that may help explain the basis of resistance of some cancers to proteasome inhibitors and underline the need for usage of PIs in combination with traditional chemotherapy.     

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.     

细菌蛋白酶体及蛋白酶体抑制剂研究进展

蛋白酶体在真核生物、古菌和部分细菌(主要是放线菌)的胞内蛋白质降解中起着至关重要的作用。虽然三域生物蛋白酶体的结构相似,但细菌蛋白酶体在组装、调节、生理功能等方面与真核生物和古菌都截然不同。研究细菌蛋白酶体不仅有助于认识其起源和进化历程,也将为发掘蛋白酶体抑制剂(proteasome inhibitor, PI)这类具有广阔药用前景的化合物提供指导。本文综述了细菌蛋白酶体的结构、功能和进化假说,并概括了细菌蛋白酶体抑制剂的最新研究进展,期望为相关研究提供参考。