JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

Cloning and Characterization of Novel CIS Family Genes

We have reported two JAK-signaling modulators, CIS (cytokine-inducible SH2 protein) and JAB (JAK2 binding protein), which are structurally related. Here we cloned three additional CIS family genes (CIS2, CIS3, and CIS4) on the basis of an expression sequence tag (EST) database search. We also found at least two additional candidates of this gene family in the database. These genes were induced by erythropoietin and granulocyte-macrophage colony stimulating factor in certain hematopoietic cell lines. The SH2 domain and a C-terminal 40 amino acid region, designated the CIS homology domain (CH domain), are highly conserved in this family, while the N-terminal regions of these proteins share little similarity. A yeast two-hybrid assay andin vitroandin vivobinding assays revealed that in addition to JAB, CIS3 bound to the JAK2 tyrosine kinase domain (JH1), although the interaction of CIS3 with the JAK2-JH1 domain was much weaker than that of JAB. Transient expression of JAB and CIS3, but not other CISs, strongly inhibited leukemia inhibitory factor (LIF)-induced STAT3-reporter gene activation in 293 cells. Furthermore, constitutive overexpression of JAB and CIS3 in M1 leukemia cells prevented LIF-induced differentiation and growth arrest. Although the physiological function remains to be investigated, CIS family genes could play a role in the negative regulation of cytokine signaling by interacting with specific targets.     

The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers

The human “protein interacting with carboxyl terminus 1” (PICT-1), also designated as the “glioma tumor suppressor candidate region 2 gene product”, GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.     

Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif

Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.     

Perk Ablation Ameliorates Myelination in S63del-Charcot–Marie–Tooth 1B Neuropathy

In peripheral nerves, P0 glycoprotein accounts for more than 20% of myelin protein content. P0 is synthesized by Schwann cells, processed in the endoplasmic reticulum (ER) and enters the secretory pathway. However, the mutant P0 with S63 deleted (P0S63del) accumulates in the ER lumen and induces a demyelinating neuropathy in Charcot–Marie–Tooth disease type 1B (CMT1B)–S63del mice. Accumulation of P0S63del in the ER triggers a persistent unfolded protein response. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER stress sensor that phosphorylates eukaryotic initiation factor 2 alpha (eIF2alpha) in order to attenuate protein synthesis. We have shown that increasing phosphophorylated-eIF2alpha (P-eIF2alpha) is a potent therapeutic strategy, improving myelination and motor function in S63del mice. Here, we explore the converse experiment: Perk haploinsufficiency reduces P-eIF2alpha in S63del nerves as expected, but surprisingly, ameliorates, rather than worsens S63del neuropathy. Motor performance and myelin abnormalities improved in S63del//Perk+/− compared with S63del mice. These data suggest that mechanisms other than protein translation might be involved in CMT1B/S63del neuropathy. In addition, Perk deficiency in other cells may contribute to demyelination in a non–Schwann-cell autonomous manner.     

RBBP6 interacts with multifunctional protein YB-1 through its RING finger domain, leading to ubiquitination and proteosomal degradation of YB-1

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as a putative E3 ubiquitin ligase due to the presence of a RING finger domain, although no substrate has been identified up to now. Using the RING finger domain as bait in a yeast two-hybrid screen, we identified YB-1 (Y-box binding protein 1) as a binding partner of RBBP6, localising the interaction to the last 62 residues of YB-1. We showed, furthermore, that both full-length RBBP6 and the isolated RING finger domain were able to ubiquitinate YB-1, resulting in its degradation in the proteosome. As a result, RBBP6 was able to suppress the levels of YB-1 in vivo and to reduce its transactivational ability. In the light of the important role that YB-1 appears to play in tumourigenesis, our results suggest that RBBP6 may be a relevant target for therapeutic drugs aimed at modifying the activity of YB-1.     

Anticancer compound Oplopantriol A kills cancer cells through inducing ER stress and BH3 proteins Bim and Noxa

Oplopantriol-A (OPT) is a natural polyyne from Oplopanax horridus. We show here that OPT preferentially kills cancer cells and inhibits tumor growth. We demonstrate that OPT-induced cancer cell death is mediated by excessive endoplasmic reticulum (ER) stress. Decreasing the level of ER stress either by inactivating components of the unfolded protein response (UPR) pathway or by expression of ER chaperone protein glucose-regulated protein 78 (GRP78) decreases OPT-induced cell death. We show that OPT induces the accumulation of ubiquitinated proteins and the stabilization of unstable proteins, suggesting that OPT functions, at least in part, through interfering with the ubiquitin/proteasome pathway. In support of this, inhibition of protein synthesis significantly decreased the accumulation of ubiquitinated proteins, which is correlated with significantly decreased OPT-induced ER stress and cell death. Finally, we show that OPT treatment significantly induced the expression of BH3-only proteins, Noxa and Bim. Knockdown of both Noxa and Bim significantly blocked OPT-induced cell death. Taken together, our results suggest that OPT is a potential new anticancer agent that induces cancer cell death through inducing ER stress and BH3 proteins Noxa and Bim.