Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells

Constitutive NF-kappaB activity has emerged as an important cell survival regulator. Canonical inducible NF-kappaB activation involves IkappaB kinase (IKK)-dependent dual phosphorylation of Ser 32 and 36 of IkappaBalpha to cause its beta-TrCP-dependent ubiquitylation and proteasomal degradation. We recently reported that constitutive NF-kappaB (p50/c-Rel) activity in WEHI231 B cells is maintained through proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in a manner that requires Ser 32 and 36, without the requirement of a direct interaction with beta-TrCP. Here we specifically examined whether dual phosphorylation of Ser 32 and 36 was required for PIR degradation. Through mutagenesis studies, we found that dual replacement of Ser 32 and 36 with Glu permitted beta-TrCP and proteasome-dependent, but not PIR, degradation. Moreover, single replacement of either Ser residue with Leu permitted PIR degradation in WEHI231 B cells. These results indicate that PIR degradation occurs in the absence of dual phosphorylation, thereby explaining the beta-TrCP-independent nature of the PIR pathway. Additionally, we found evidence that PIR IkappaBalpha degradation controls constitutive NF-kappaB activation in certain multiple myeloma cells. These results suggest that B lineage cells can differentiate between PIR and canonical IkappaBalpha degradation through the absence or presence of dually phosphorylated IkappaBalpha.     

Phosphorylated IkappaBalpha is a component of Lewy body of Parkinson's disease

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.     

Assembly of the 20S proteasome

The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

The heat-shock-induced suppression of the IkappaB/NF-kappaB cascade is due to inactivation of upstream regulators of IkappaBalpha through insolubilization

Heat shock (HS) was found to suppress the IkappaB/NF-kappaB cascade via the inhibition of IkappaB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-alpha-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IkappaBalpha degradation. Upstream activators of IKK, such as NF-kappaB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-alpha-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IkappaB/NF-kappaB pathway.     

Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1–60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H₂O₂ resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H₂O₂ of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.     

Synergistic interaction of proteasome and topoisomerase II inhibition in multiple myeloma

Multiple myeloma is a malignancy of terminally differentiated plasma cells and is incurable in the majority of the patients. Thus, novel effective treatment regimens are urgently needed. In this study, we examined the effects of co-treatment with proteasome-inhibitor bortezomib and topoisomerase II inhibitor etoposide in multiple myeloma cells lines OPM-2, RPMI-S and NCI-H929. Using the median effect method of Chou and Talalay, we evaluated the combination indices (CI) for simultaneous and sequential treatment schedules. In the sequential treatment schedule, we found strong synergistic effects in all three cell lines, even at low single-agent cytotoxicity levels. When cells were treated simultaneously with both drugs, the synergy was present but less pronounced than in the sequential treatment schedule. The synergistic effects observed in the co-treatment schedules were accompanied by an inhibition of anti-apoptotic effects that were induced by etoposide alone. Namely, bortezomib abrogated both etoposide-induced NF-κB activation and etoposide-induced bcl-2 up-regulation. Our data suggest that combining etoposide with bortezomib might be useful for cancer treatment, as bortezomib potentially inhibits counter-regulatory mechanisms of tumor cells, which are induced by topoisomerase II inhibition and which may contribute to acquired chemoresistance.     

Selective degradation of oxidatively modified protein substrates by the proteasome

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are recognized by the 20S 'core' proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hydrophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates during diseases and the aging process.     

NF-κB、IκB、IKK在非小细胞肺癌中的表达及意义

目的检测非小细胞肺癌(non-small cell lung cancer,NSCLC)中NF-κB P65、p-IκBα(IκBα磷酸化)、p-IKKβ(IKKβ磷酸化)的表达情况及其与NSCLC临床特征的关系。方法采用免疫组化Elivision法检测NF-κB P65、p-IκBα、p-KKβ在56例NSCLC中表达情况,以20例癌旁组织作为对照。结果在NSCLC中NF-κB P65、p-IκBα、p-IKKβ的表达阳性率分别为83.9%(47/56)、55.7%(31/56)、69.6%(39/56),癌旁组织三者分别为20%(4/20)、25%(5/20)、30%(6/20),NF-κB P65、p-IκBα、p-IKKβ的表达与吸烟史、TNM分期、淋巴结转移相关,差异有统计学意义(P<0.05)。结论 NF-κB P65、p-IκBα、p-IKKβ高表达与NSCLC的发生、发展起着重要作用。     

Mustard NPR1, a mammalian IκB homologue inhibits NF-κB activation in human GBM cell lines

NF-κB activity is tightly regulated by IκB class of proteins. IκB proteins possess ankyrin repeats for binding to and inhibiting NF-κB. The regulatory protein, NPR1 from Brassica juncea possesses ankyrin repeats with sequence similarity to IκBα subgroup. Therefore, we examined whether stably expressed BjNPR1 could function as IκB in inhibiting NF-κB in human glioblastoma cell lines. We observed that BjNPR1 bound to NF-κB and inhibited its nuclear translocation. Further, BjNPR1 expression down-regulated the NF-κB target genes iNOS, Cox-2, c-Myc and cyclin D1 and reduced the proliferation rate of U373 cells. Finally, BjNPR1 decreased the levels of pERK, pJNK and PKCα and increased the Caspase-3 and Caspase-8 activities. These results suggested that inhibition of NF-κB activation by BjNPR1 can be a promising therapy in NF-κB dependent pathologies.     

Potentiation of humoral immune response and activation of NF-kappaB pathway in lymphocytes in experimentally induced hyperthyroid rats

This study explored the effect of hyperthyroid state on humoral immune response and NF-kappaB signaling in lymphocytes. Male Wistar rats were treated with l-thyroxin for four weeks. Animals were immunized with sheep red blood cells (SRBC) after three weeks of l-thyroxin treatment. After one week of immunization, serum anti-SRBC titer was measured and NF-kappaB signaling was studied in lymphocytes by Western blot analysis of p-IKB-alpha, IKB-alpha, and p65. These results were compared with that of control rats. Antibody response and density of p-IKB-alpha and p65 were significantly higher in l-thyroxin treated rats in comparison to controls. The antibody response was found to have significant correlation with density of p-IKB-alpha and p65. Our results indicate that NF-kappaB signaling pathway in lymphocytes is activated in hyperthyroid state which might play a role in potentiation of antibody response.