Curcusone D,a novel ubiquitin–proteasome pathway inhibitor via ROS-induced DUB inhibition,is synergistic with bortezomib against multiple myeloma cell growth

Background

Ubiquitin–proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM).

Methods

Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H₂DCFDA.

Results

Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells.

Conclusions

Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib.

General significance

The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.     

The inhibitor peptide of the mitochondrial F1 · F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F₁ · F₀-ATPase has been studied. [^125I]Iodoazidocalmodulin binds to the ε-subunit and to the endogeneous ATPase inhibitor peptide in a Ca²⁺-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca²⁺-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca²⁺-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

The nitric oxide donor sodium nitroprusside requires the 18?kDa Translocator Protein to induce cell death

Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, β-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.     

Comparison of the TCR ζ-chain with the FcR γ-chain in chimeric TCR constructs for T cell activation and apoptosis

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

The novel endosomal membrane protein Ema interacts with the class C Vps–HOPS complex to promote endosomal maturation

Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps–HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps–HOPS complex function.     

Ezrin interacts with L-periaxin by the “head to head and tail to tail” mode and influences the location of L-periaxin in Schwann cell RSC96

In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.     

The β1 subunit of Na/K-ATPase interacts with BKCa channels and affects their steady-state expression on the cell surface

Large conductance Ca²⁺-activated K⁺ channels (BK_Ca) encoded by the Slo1 gene play a role in the physiological regulation of many cell types. Here, we show that the β1 subunit of Na⁺/K⁺-ATPase (NKβ1) interacts with the cytoplasmic COOH-terminal region of Slo1 proteins. Reduced expression of endogenous NKβ1 markedly inhibits evoked BK_Ca currents with no apparent effect on their gating. In addition, NKβ1 down-regulated cells show decreased density of Slo1 subunits on the cell surface.

Structured summary

MINT-7260438, MINT-7260555: Slo1 (uniprotkb:Q8AYS8) physically interacts (MI:0915) with NKbeta1 (uniprotkb:P08251) by anti bait coimmunoprecipitation (MI:0006)MINT-7260587, MINT-7260606, MINT-7260619, MINT-7260632: Slo1 (uniprotkb:Q08460) physically interacts (MI:0915) with NKbeta 1 (uniprotkb:P08251) by pull down (MI:0416)MINT-7260570: NKbeta1 (uniprotkb:P08251) and Slo1 (uniprotkb:Q8AYS8) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7260414: Slo1 (uniprotkb:Q08460) physically interacts (MI:0915) with NKbeta1 (uniprotkb:P08251) by two hybrid (MI:0018)     

The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.     

The �æ� variant of T cell large granular lymphocyte leukemia is very similar to the common ���� type: report of two cases

The vast majority of cases of T cell large granular lymphocyte (T-LGL) leukemia have a CD3+, CD4−, CD8+ phenotype and express the αβ T cell receptor. Whether the rare γδ variant should be included in the same diagnostic category is currently unclear. Two well-characterized cases of γδ T-LGL leukemia were identified by our laboratory in 2007. These two cases and other reports of γδ T-LGL leukemia were compared with the common αβ variant. Other than more often being negative for both CD4 and CD8 (in about 35% to 40% of cases), the γδ variant of T-LGL leukemia is similar to the common αβ type in virtually all respects and should be included in the general category of T-LGL leukemia. However, it is important to exclude other more aggressive γδ T cell lymphoproliferative disorders.     

The rate-limiting step of sulfiredoxin is associated with the transfer of the γ-phosphate of ATP to the sulfinic acid of overoxidized typical 2-Cys peroxiredoxins

The eukaryotic sulfiredoxin (Srx) catalyzes the reduction of overoxidized typical 2-Cys peroxiredoxins PrxSO₂ via ATP/Mg²⁺-dependent phosphorylation of the sulfinic acid group, followed by formation of a PrxSO-SSrx thiolsulfinate intermediate. Using real-time kinetics of wild-type and C84A Srxs, and pH-rate profiles with ATP/Mg²⁺ analogues, we show that the rate-limiting step of the reaction is associated with the chemical process of transfer of the γ-phosphate of ATP to the sulfinic acid, in contrast to that described by Jönsson et al. [7]. Two pK_apps of 6.2 and 7.5 were extracted from the bell-shaped pH-rate profile, corresponding to the γ-phosphate of ATP, and to an acid–base catalyst, respectively.