Solution structure of the ubiquitin-like domain of human DC-UbP from dendritic cells

The previously identified dendritic cell-derived ubiquitin-like protein (DC-UbP) was implicated in cellular differentiation and apoptosis. Sequence alignment suggested that it contains a ubiquitin-like (UbL) domain in the C terminus. Here, we present the solution NMR structure and backbone dynamics of the UbL domain of DC-UbP. The overall structure of the domain is very similar to that of Ub despite low similarity (<30%) in amino-acid sequence. One distinct feature of the domain structure is its highly positively charged surface that is different from the corresponding surfaces of the well-known UbL modifiers, Ub, NEDD8, and SUMO-1. The key amino-acid residues responsible for guiding polyubiquitinated proteins to proteasome degradation in Ub are not conserved in the UbL domain. This implies that the UbL domain of DC-UbP may have its own specific interaction partners with other yet unknown cellular functions related to the Ub pathway.     

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.     

Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family.

Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFbeta in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells. In this study, we purified a 33.5 kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-(Ubi-L) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon gamma-activated D.10 cells. The 33.5 kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.     

Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain

The sequence of the ubiquitin protein is highly conserved between species and has facilitated the cloning of numerous ubiquitin-like proteins. In the present study, we report the cloning of the cDNA for human ubiquilin 3 (UBQLN3). The deduced amino acid sequence of UBQLN3 contains a UBQ domain (ubiquitin-like) in the amino terminus as well as two highly conserved domains found in several recently cloned ubiquitin-like proteins. One of these domains, termed the NP domain, is a highly conserved 93 amino acid region present in UBQLN3 and several ubiquitin-like proteins. The last conserved domain is the UBA domain (ubiquitin-associated) found in a variety of proteins of the ubiquination pathway. The human UBQLN3 gene was mapped to the 11p15 region of chromosome 11. Northern blot analysis of multiple human and mouse tissues demonstrated UBQLN3 mRNA expression specifically in testis.     

A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins are known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin-activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins. Ubiquitin-activating enzyme E1-domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin-like protein, ubiquitin-fold modifier 1 (Ufm1), by forming a high-energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin-like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.     

Mimosine induces apoptosis in the HL60 human tumor cell line

Mimosine, a plant amino acid not found in proteins, has been widely used as a synchronizing agent, blocking the progression of cell cycle on the G1/S phase border. The mechanism by which this block is achieved is still unclear. We report that in HL60 cells the synchronization is related to an increase in apoptosis. Another human tumor cell line, K562, is insensitive to both phenomena thereby demonstrating that apoptosis observed in HL60 is line-specific. We hypothesize that the mimosine-induced apoptosis and alteration of the cell cycle is due to the inhibition of hypusine generation.     

人白血病细胞系P2X7受体的表达及其介导的细胞内钙浓度变化

采用半定量RT-PCR和流式细胞术,在基因和蛋白水平研究了白血病细胞系U937、HL60和Ramos细胞P2X7受体的表达。荧光染料Fura-2／AM负载后,用荧光分光光度计测定P2X7受体激动剂三磷酸腺苷(adenosine 5′-triphosphate,ATP)和苯甲酰苯甲酸ATP(2′,3′-O-(4-benzoyl)benzoyl-ATP,BzATP)刺激前后细胞内钙离子浓度的变化,以确认其功能。结果表明：U937和HL60细胞系表达P2X7受体的mRNA和蛋白,Ramos不表达;在激动剂的刺激下,可引发U937和HL60细胞胞内钙浓度的显著升高,但对Ramos没有作用。当去除胞外钙离子时,ATP和BzATP刺激均不能引起U937和HL60细胞胞内钙离子浓度的升高。提示U937和HL60细胞表达P2X7受体的基因和功能蛋白,Ramos细胞则不表达该受体。     

ThiS可能作为一个原核翻译后修饰分子通过二硫键结合细胞蛋白

泛素及其相关蛋白是真核细胞中广泛存在的结构高度保守的一类小分子蛋白,参与蛋白翻译后修饰. 尽管在少数原核种属含有Pupylation这样的翻译后修饰,在原核细胞中尚未发现通用的泛素样修饰系统. ThiS是原核细胞广泛存在的泛素样小蛋白分子,它作为硫转运蛋白参与辅助因子的合成. 当与靶蛋白融合重组表达时,ThiS可降低靶蛋白在大肠杆菌中的稳定性. 本研究旨在探讨ThiS是否可能在原核细胞中参与翻译后修饰. ThiS在大肠杆菌中重组表达时,它可与细胞蛋白游离巯基发生共价结合,但与真核细胞泛素修饰不同,ThiS是通过12位半胱氨酸的游离巯基与蛋白形成二硫键,而不是通过C端活化的硫代羧基的转化过程发生共价结合. 在细胞内,氧化应激可诱导ThiS与蛋白的共价结合. 结果提示,ThiS在大肠杆菌中与细胞蛋白的结合,可能与真核细胞泛素化修饰在功能上存在进化联系;原核细胞中这种ThiS的结合形式可能代表一种古老的原核泛素样修饰方式.     

Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex

Sentrin-1/SUMO-1 is a novel ubiquitin-like protein, which can covalently modify a limited number of cellular proteins. Here we report the identification of the sentrin-activating enzyme complex, which consists of two proteins AOS1 and UBA2. Human AOS1 is homologous to the N-terminal half of E1, whereas human UBA2 is homologous to the C-terminal half of E1. The human UBA2 gene is located on chromosome 19q12. Human UBA2 could form a beta-mercaptoethanol-sensitive conjugate with members of the sentrin family, but not with ubiquitin of NEDD8, in the presence of AOS1. Identification of human UBA2 and AOS1 should allow a more detailed analysis of the enzymology of the activation of ubiquitin-like proteins.     

FAT10, a ubiquitin-independent signal for proteasomal degradation

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.     

Promoter regulatory elements and DNase I-hypersensitive sites involved in serglycin proteoglycan gene expression in human erythroleukemia, CHRF 288-11, and HL-60 cells

We have compared regulation of the serglycin gene in human erythroleukemia (HEL) and CHRF 288-11 cells, which have megakaryocytic characteristics, with promyelocytic HL-60 cells. Deletion constructs were prepared from the region -1123/+42 to -20/+42, and putative regulatory sites were mutated. In all three cell lines, the two major regulatory elements for constitutive expression were the (-80)ets site and the cyclic AMP response element (CRE) half-site at -70. A protein from HEL and CHRF, but not HL60, nuclear extracts bound to the (-80)ets site. Another protein from all three cell lines bound to the (-70)CRE. Phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (dbcAMP) increased expression of the reporter in HEL cells 2.5-3- and 4.5-fold, respectively, from all constructs except those with (-70)CRE mutations. PMA virtually eliminated expression of serglycin mRNA and promoter constructs, but dbcAMP increased expression in HL-60 cells. The effects of PMA and dbcAMP on promoter expression correlated with mRNA expression. The strengths of two DNase I-hypersensitive sites in the 5'-flanking region and the first intron in all three cells correlated with relative endogenous serglycin mRNA expression. An additional DNase I-hypersensitive site in HL60 DNA in the first intron may be related to the high serglycin expression in HL60 relative to HEL or CHRF cells.     

Ubiquitin and its kin: how close are the family ties?

Modification of proteins by the covalent attachment of ubiquitin is known to target them for degradation by proteasomes. Several proteins have been discovered recently that are related to ubiquitin or function similarly. Some of these proteins act as modifiers; others bear ubiquitin-like domains embedded in their polypeptide chain but do not form conjugates with cellular proteins. Ubiquitin-like proteins mediate an impressive range of cellular functions, including cell-cycle progression, DNA repair and apoptosis. Recent discoveries endorse the view that, in many cases, the function of the relatives of ubiquitin is linked to the ubiquitin pathway.