Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1

SRC-3/AIB1 is a steroid receptor coactivator with potent growth-promoting activity, and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue-rich region in SRC-3 is an important determinant for aPKC-mediated phosphorylation and stabilization. The mechanism of the aPKC-mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.     

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.     

Ubiquitin-dependent and -independent proteasomal degradation of apoB associated with endoplasmic reticulum and Golgi apparatus,respectively, in HepG2 cells

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin-dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi-associated apoB was not ubiquitinated, indicating an ubiquitin-independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER-associated proteasomal degradation of apoB in the ER is ubiquitin-dependent whereas that occurring in the Golgi is ubiquitin-independent.     

Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how alpha-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated alpha-synuclein strongly inhibited the function of the 26 S proteasome. The IC(50) of aggregated alpha-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nm. Aggregated alpha-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nm. In contrast, the IC(50) of aggregated alpha-synuclein for 20 S proteasomal activity was > 1 microm. This suggests that aggregated alpha-synuclein selectively interacts with the 19 S cap. Monomeric alpha-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric alpha-synuclein inhibited the activity of the 20 S proteasomal core with an IC(50) > 10 microm, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 microm, and exhibited only partial inhibition (50%) of the 26 S ubiquitin-independent proteasomal activity at doses up to 10 mm. Binding studies demonstrate that both aggregated and monomeric alpha-synuclein selectively bind to the proteasomal protein S6', a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated alpha-synuclein could be mediated by interaction with S6'.     

beta-Synuclein reduces proteasomal inhibition by alpha-synuclein but not gamma-synuclein

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.     

alpha-Synuclein protofibrils inhibit 26 S proteasome-mediated protein degradation: understanding the cytotoxicity of protein protofibrils in neurodegenerative disease pathogenesis

The impaired ubiquitin-proteasome activity is believed to be one of the leading factors that contribute to Parkinson disease pathogenesis partially by causing alpha-synuclein aggregation. However, the relationship between alpha-synuclein aggregation and the impaired proteasome activity is yet unclear. In this study, we examined the effects of three soluble alpha-synuclein species (monomer, dimer, and protofibrils) on the degradation activity of the 26 S proteasome by reconstitution of proteasomal degradation using highly purified 26 S proteasomes and model substrates. We found that none of the three soluble alpha-synuclein species impaired the three distinct peptidase activities of the 26 S proteasome when using fluorogenic peptides as substrates. In striking contrast, alpha-synuclein protofibrils, but not monomer and dimer, markedly inhibited the ubiquitin-independent proteasomal degradation of unstructured proteins and ubiquitin-dependent degradation of folded proteins when present at 5-fold molar excess to the 26 S proteasome. Together these results indicate that alpha-synuclein protofibrils have a pronounced inhibitory effect on 26 S proteasome-mediated protein degradation. Because alpha-synuclein is a substrate of the proteasome, impaired proteasomal activity could further cause alpha-synuclein accumulation/aggregation, thus creating a vicious cycle and leading to Parkinson disease pathogenesis. Furthermore we found that alpha-synuclein protofibrils bound both the 26 S proteasome and substrates of the 26 S proteasome. Accordingly we propose that the inhibitory effect of alpha-synuclein protofibrils on 26 S proteasomal degradation might result from impairing substrate translocation by binding the proteasome or sequestrating proteasomal substrates by binding the substrates.     

The proteasomal system and HNE-modified proteins

Metabolic processes and environmental conditions cause the constant formation of oxidizing species over the lifetime of cells and organisms. This leads to a continuous oxidation of intracellular components, including lipids, DNA and proteins. During the extensively studied process of lipid peroxidation, several reactive low-molecular weight products are formed, including reactive aldehydes as 4-hydroxynonenal (HNE). These aldehydic lipid peroxidation products in turn are able to modify proteins. The degradation of oxidized and oxidatively modified proteins is an essential part of the oxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasomal system. The proteasomal system by itself is a multicomponent system responsible for the degradation of the majority of intracellular proteins. It has been shown that some, mildly cross-linked, HNE-modified proteins are preferentially degraded by the proteasome, but extensive modification with this cross-linking aldehyde leads to the formation of protein aggregates, that can actually inhibit the proteasome. This review summarizes our knowledge of the interactions between lipid peroxidation products, proteins, and the proteasomal system.     

Ubiquitin-independent degradation of proteins by the proteasome

The proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here.     

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by β oxidation of fatty acids in the isolated rat heart

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β-oxidation pathways. The hypothesis driving this report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1 mM octanoate). During ischemia, HNE concentrations doubled and the glutathione–HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

Proteasomal degradation of tau protein

Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.     

4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules.

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome, a process accelerated by the UBL-UBA domain protein NEDD8 ultimate buster 1-long. Here, we show that FAT10-mediated degradation occurs independently of poly-ubiquitylation as purified 26S proteasome readily degraded FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro. Interestingly, the 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock-down of NUB1L attenuated the degradation of FAT10-DHFR in intact cells suggesting that NUB1L determines the degradation rate of FAT10-linked proteins. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L-dependent targeting signal for proteasomal degradation.     

Leishmania express a functional Cdc20 homologue

Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.     

NF-kappaB dictates the degradation pathway of IkappaBalpha

IkappaB proteins are known as the regulators of NF-kappaB activity. They bind tightly to NF-kappaB dimers, until stimulus-responsive N-terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IkappaBalpha is an unstable protein whose rapid degradation is slowed upon binding to NF-kappaB, but it is not known what dynamic mechanisms control the steady-state level of total IkappaBalpha. Here, we show clearly that two degradation pathways control the level of IkappaBalpha. Free IkappaBalpha degradation is not controlled by IKK or ubiquitination but intrinsically, by the C-terminal sequence known as the PEST domain. NF-kappaB binding to IkappaBalpha masks the PEST domain from proteasomal recognition, precluding ubiquitin-independent degradation; bound IkappaBalpha then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IkappaBalpha protein; alteration of free IkappaBalpha degradation dampens NF-kappaB activation. In addition, we find that both free and bound IkappaBalpha are similar substrates for IKK, and the preferential phosphorylation of NF-kappaB-bound IkappaBalpha is due to stabilization of IkappaBalpha by NF-kappaB.     

The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.     

Multiple Sclerosis Autoantigen Myelin Basic Protein Escapes Control by Ubiquitination during Proteasomal Degradation

The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.     

Combined treatment of human multiple myeloma cells with bortezomib and doxorubicin alters the interactome of 20S proteasomes

The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy.

Abbreviations: BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.