David C.H. Yang

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Cancer Preventive Isothiocyanates Induce Selective Degradation of Cellular α- and β-Tubulins by Proteasomes

Although it is conceivable that cancer preventive isothiocyanates (ITCs), a family of compounds in cruciferous vegetables, induce cell cycle arrest and apoptosis through a mechanism involving oxidative stress, our study shows that binding to cellular proteins correlates with their potencies of apoptosis induction. More recently, we showed that ITCs bind selectively to tubulins. The differential binding affinities toward tubulin among benzyl isothiocyanate, phenethyl isothiocyanate, and sulforaphane correlate well with their potencies of inducing tubulin conformation changes, microtubule depolymerization, and eventual cell cycle arrest and apoptosis in human lung cancer A549 cells. These results support that tubulin binding by ITCs is an early event for cell growth inhibition. Here we demonstrate that ITCs can selectively induce degradation of both α- and β-tubulins in a variety of human cancer cell lines in a dose- and time-dependent manner. The onset of degradation, a rapid and irreversible process, is initiated by tubulin aggregation, and the degradation is proteasome-dependent. Results indicate that the degradation is triggered by ITC binding to tubulin and is irrelevant to oxidative stress. This is the first report that tubulin, a stable and abundant cytoskeleton protein required for cell cycle progression, can be selectively degraded by a small molecule.

Expression of SUMO-2/3 Induced Senescence through p53- and pRB-mediated Pathways

Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3ΔGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3ΔGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

ABSTRACT An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitro-anilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 μM. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

Mammalian aminoacyl-tRNA synthetases

Publisher Summary This chapter discusses the recent developments of the studies on the structure-function relationship of mammalian synthetases, with emphasis on those features that are unique in mammalian synthetases and are for the most part absent in bacterial and yeast enzymes. Translation and transcription constitute the two major steps of gene expression in all organisms. Aminoacyl-tRNA synthetases (RSs) carry out the key role of the interpretation of the genetic code by the covalent attachment of specific amino acids to cognate tRNAs. The chapter discusses various classifications of mammalian aminoacyl-tRNA synthetases with several examples. The chapter also discusses the general structure of the RS complex dissociation and organization of the synthetases complex primary structures of mammalian synthetases. The chapter describes several distinct characteristics of N-terminal extensions in mammalian aminoacyl-tRNA synthetases. Mammalian RSs are obviously more complex than their bacterial or yeast counterparts. The structure and function of mammalian RSs are not well understood despite the tremendous progress that has been made at an increasingly rapid pace. The occurrence of synthetases complexes provides excellent models for the elucidation of basic principles in molecular interactions for highly complex machinery that requires efficiency, fidelity, and versatility. Synthetases are critically important in the interpretation of the genetic code and are responsible for the biosynthesis of all proteins in all cells and tissues. In view of the physiological roles of amino acids, elucidation of such relation is obviously important. The chapter concludes with the organization of synthetases and protein biosynthetic machinery.

High molecular mass amino acyl-tRNA synthetase complexes in eukaryotes

Aminoacyl-t RNA synthetases (AARS) are enzymes which play an indispensable role in protein biosynthe- sis by catalyzing the formation of aminoacyl-tRNA from amino acid, the cognate tRNA, and ATP by highly selective intermolecular interactions [57]. Joachimiak and Barciszewski [41] have provided an extensive compilation of the properties of the amino- acyl-tRNA synthetases; however, information on the eukaryotic highM r (HMr) complexes of aminoacyl-tRNA synthetases was lacking. Here, we intend to fill this void by providing a summary of the properties of the eukaryotic aminoacyl-tRNA synthetase complexes. Eukaryotic aminoacyl-tRNA synthetases may occur as complexes with Mr-values of >106 in contrast to the prokaryotic counterparts which have Mr-values of <250 000. These eukaryotic HMr--AARS complexes appear ubiquitous in a wide spectrum of cell types from yeast to human placenta as shown in table 1. Although not all 20 aminoacyl-tRNA synthetases were examined in each case shown in table 1, it appears that the AARS commonly associated with M r complexes are those specific for Arg, Gin, Glu, fie, Leu, Lys and Met. The properties of these HMr-AARS complexes are most consistent with multienzyme complexes of aminoacyl-tRNA synthetases [ 19,20,43, 46]. The physicochemical properties, composition, and stoichiometry of the more rigorously character- ized complexes are shown in table 2. The mechanism(s) of intermolecular interaction between the aminoacyl-tRNA synthetases is not known, but the putative interactions of aminoacyl- tRNA synthetases with a variety of biomolecules have been suggested to play a role in complex formation as shown in table 3. Our present knowledge of the func- * To whom correspondences should be addressed tional significance of HMr--AARS is profoundly lack- ing; however, interactions of the aminoacyl-tRNA synthetases with other components of the protein biosynthetic machinery and other enzymes suggest the intriguing possibility of higher organization of eukaryotic protein biosynthesis. Table 4 is a summary of the possible interactions of the aminoacyl-tRNA synthetases with subcellular components and other enzymes. This presentation is a brief summary of the prop- erties of the high molecular weight eukaryotic amino- acyl-tRNA synthetase complexes. We hope that this compilation will complement that presented in [41 ] and will provide useful information for workers in this and other related fields.

Application of the continuous variation method to cooperative interactions: mechanism of Fe(II)-ferrozine chelation and conditions leading to anomalous binding ratios.

The method of continuous variation, often known as the Job plot, has long been used for determining the stoichiometry of two interacting components. The correct binding ratio, n, is generally obtained when the total concentration of the reactants, C(o), is much greater than the dissociation constants involved. For non-cooperative binding systems, the stoichiometry varies between one and n as C(o) increases; whereas for positive cooperative systems, values larger than n may be observed at low C(o). In this report, we present examples to illustrate how the changing apparent stoichiometries as a function of C(o) can provide clues for differentiating various binding mechanisms. To test these concepts, we examined the chelation of Fe(II) with ferrozine in the range of C(o)=7 to 210 microM with Fe(II) expressed in molar concentration or in terms of its binding equivalents (three in this case). The results were analyzed according to several models and found to be most consistent with the mechanism of one-step complex formation or infinite cooperativity with a K(d) of 8 microM.

Polyubiquitylation of PARP‐1 through ubiquitin K48 is modulated by activated DNA, NAD+, and dipeptides

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is the most abundant and the best‐studied isoform of a family of enzymes that catalyze the polymerization of ADP‐ribose from NAD+ onto target proteins. PARP‐1 is well known to involve in DNA repair, genomic stability maintenance, transcription regulation, apoptosis, and necrosis. Polyubiquitylation targets proteins towards degradation and regulates cell cycle progression, transcription, and apoptosis. Here we report polyubiquitylation of PARP‐1 in mouse fibroblasts in the presence of proteasome inhibitor and in full‐length recombinant PARP‐1 in vitro under standard ubiquitylation assay conditions by immunoprecipitation and immunoblotting. Mutation of ubiquitin K48R but not ubiquitin K63R abolishes polyubiquitylation of PARP‐1, indicating that K48 of ubiquitin was used in the formation of polyubiquitin chain and that ubiquitylated PARP‐1 is likely destined for degradation. Full‐length PARP‐1 was ubiquitylated most likely at the N‐terminal 24 kDa domain of PARP‐1 as suggested by the inhibition of ubiquitylation by activated DNA and the absence of polyubiquitin in the C‐terminal 89 kDa PARP‐1 derived from caspase‐catalyzed cleavage. NAD+ inhibited ubiquitylation of PARP‐1, while dipeptides ArgAla and LeuAla enhanced ubiquitylation of PARP‐1. ATP inhibited the synthesis of poly(ADP‐ribose) by PARP‐1 and affinity purified polyubiquitylated PARP‐1 was active in PAR synthesis. The results suggest polyubiquitylation of PARP‐1 could regulate poly(ADP‐ribosyl)ation of nuclear proteins by PARP‐1 and consequently apoptosis and PARP‐1 regulated cellular processes through ubiquitin‐dependent degradation pathways. J. Cell. Biochem. 104: 318–328, 2008.

Rat liver histidyl-tRNA synthetase. Purification and inhibition by the myositis-specific anti-Jo-1 autoantibody.

Myositis is an autoimmune inflammatory muscle disease of unknown etiology. We demonstrate directly that the antigen to the myositis-specific anti-Jo-1 antibody is histidyl-tRNA synthetase. The anti-Jo-1 antibody inhibits human HeLa and rat liver histidyl-tRNA synthetase. Using conventional and immunoaffinity chromatography with immobilized anti-Jo-1 antibody, we have purified rat liver histidyl-tRNA synthetase which has a subunit Mr 64,000 and an estimated native Mr suggesting an alpha 2 structure. The evidence indicates that the Jo-1 antigen is histidyl-tRNA synthetase, and that some of the histidyl-tRNA synthetase structure are conserved across species.

FAT10 modifies p53 and upregulates its transcriptional activity

FAT10, also known as diubiquitin, has been implicated in the regulation of diverse cellular processes, including mitosis, immune response, and apoptosis. We seek to identify FAT10-targeted proteins, an essential step in elucidating the physiological function of FAT10. To this end, human FAT10 or its non-conjugatable derivative, FAT10ΔGG, was overexpressed in HEK293 cells. We observed a number of high molecular weight FAT10 conjugates in cells expressing wild-type FAT10, but not in FAT10ΔGG. The FAT10 conjugates are inducible by TNF-α and accumulated significantly when cells were treated with proteasome inhibitor, MG132. Among them, tumor suppressor p53 was found to be FATylated. The p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells. In addition, overexpressing FAT10 in HEK293 cells also reduced the population of p53 which cross reacted with monoclonal anti-p53 antibody, PAB240, known to recognize only the transcriptionally inactive p53. FAT10 in the nucleus was found co-localized with p53 and altered its subcellular compartmentalization. Furthermore, overexpressing FAT10 led to a reduction in the size of promyelocytic leukemia nuclear bodies (PML-NBs) and altered their distribution in the nucleus. Based on these observations, a potential mechanism which correlates FATylation of p53 to its translocation and transcriptional activation is discussed.