ABSTRACT
Interferon-stimulated gene 15 (ISG15) encodes a protein that is most upregulated by type I interferon stimulation and, upon activation, is conjugated to various target proteins in a process known as ISGylation. ISGylation has been shown to have roles in various biological phenomena such as viral infection and cancer. To gain further insight into the function of ISGylation, it would be useful to be able to identify ISGylated proteins. Here, we describe a method for the identification of proteins modified with ISG15. This method involves the generation of stable ISG15-transfectant cells, followed by affinity purification, and then identification of the ISGylated proteins by mass spectrometry.
Subject(s)
Cytokines/metabolism , Ubiquitins/metabolism , Animals , Cytokines/chemistry , Cytokines/genetics , Cytokines/isolation & purification , Humans , Mice , Peptides/chemistry , Peptides/metabolism , Protein Processing, Post-Translational , RAW 264.7 Cells , Recombinant Fusion Proteins , Ubiquitins/chemistry , Ubiquitins/genetics , Ubiquitins/isolation & purificationABSTRACT
Two new strongylophorine derivatives, along with six known strongylophorines, were isolated from the marine sponge Petrosia corticata as proteasome inhibitors. Of these, a hemiacetal mixture of strongylophorines-13/-14 was the strongest inhibitor of the proteasome with an IC50 of 2.1µM.
Subject(s)
Diterpenes/chemistry , Diterpenes/pharmacology , Petrosia/chemistry , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Diterpenes/isolation & purification , Drug Evaluation, Preclinical , Humans , Magnetic Resonance Spectroscopy , Proteasome Inhibitors/isolation & purification , Spectrometry, Mass, Fast Atom Bombardment , Structure-Activity RelationshipABSTRACT
Interferon-stimulated gene 15 kDa (ISG15) is a protein upregulated by interferon-ß that negatively regulates osteoclastogenesis. We investigated the role of ISG15 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells. RANKL stimulation induced ISG15 expression in RAW264 cells at both the mRNA and protein levels. Overexpression of ISG15 in RAW264 cells resulted in suppression of cell fusion in RANKL-stimulated cells as well as the reduced expression of ATP6v0d2, a gene essential for cell fusion in osteoclastogenic differentiation. These results suggest that ISG15 suppresses RANKL-induced osteoclastogenesis, at least in part, through inhibition of ATP6v0d2 expression.
Subject(s)
Cell Differentiation , Cytokines/metabolism , Interferon-beta/metabolism , Osteoclasts/physiology , RANK Ligand/metabolism , Animals , Bone Marrow Cells , Bone Resorption/metabolism , Cell Line , Cytokines/genetics , Macrophages/metabolism , Mice , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , RNA, Messenger/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
Halenaquinone was isolated from the marine sponge Petrosia alfiani as an inhibitor of osteoclastogenic differentiation of murine RAW264 cells. It inhibited the RANKL (receptor activator of nuclear factor-κB ligand)-induced upregulation of TRAP (tartrate-resistant acid phosphatase) activity as well as the formation of multinuclear osteoclasts. In addition, halenaquinone substantially suppressed RANKL-induced IκB degradation and Akt phosphorylation. Thus, these results suggest that halenaquinone inhibits RANKL-induced osteoclastogenesis at least by suppressing the NF-κB and Akt signaling pathways.
Subject(s)
Quinones/chemistry , Animals , Cell Differentiation/drug effects , Cell Line , I-kappa B Proteins/metabolism , Mice , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Petrosia/chemistry , Petrosia/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Quinones/isolation & purification , Quinones/pharmacology , RANK Ligand/toxicity , Signal Transduction/drug effectsABSTRACT
Two new manzamine alkaloids, acantholactam (3) and pre-neo-kauluamine (4), were isolated from the marine sponge Acanthostrongylophora ingens along with manzamine A (1) and neo-kauluamine (2). Acantholactam contains a γ-lactam ring N-substituted with a (Z)-2-hexenoic acid moiety and is proposed to be biosynthetically derived from manzamine A by oxidative cleavage of the eight-membered ring. Compound 4 was converted to the dimer 2 during storage, suggesting nonenzymatic dimer formation. Among the four isolated compounds, 1, 2, and 4 showed proteasome inhibitory activity.
Subject(s)
Alkaloids/isolation & purification , Carbazoles/isolation & purification , Porifera/chemistry , Protease Inhibitors/isolation & purification , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , Carbazoles/chemistry , Carbazoles/pharmacology , Carbolines , Indonesia , Macrophages/drug effects , Marine Biology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Oceans and Seas , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacologyABSTRACT
Five new manzamine alkaloids, acanthomanzamines A-E, were isolated from the marine sponge Acanthostrongylophora ingens. Acanthomanzamines A and B are the first examples, containing a tetrahydroisoquinoline instead of a ß-carboline in manzamine-related alkaloids. Acanthomanzamine C contains a hexahydrocyclopenta[b]pyrrol-4(2H)-one ring that may be converted from an eight-membered ring in manzamine A. Acanthomanzamines D and E have an additional oxazolidine and 2-methyloxazolidine rings, respectively, which fuse to the manzamine skeleton.
Subject(s)
Alkaloids/chemical synthesis , Carbazoles/chemical synthesis , Porifera/chemistry , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , Carbazoles/chemistry , Carbazoles/pharmacology , Humans , Molecular Structure , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.
Subject(s)
Cytoplasm/metabolism , Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Cryoelectron Microscopy , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/ultrastructure , Time Factors , Time-Lapse Imaging/methods , Red Fluorescent ProteinABSTRACT
Two new ß-carboline alkaloids, variabines A (1) and B (2), were isolated from the Indonesian marine sponge Luffariella variabilis. Their structures were elucidated from spectral data, and 1 was found to be a sulfonated derivative of 2. Although numerous ß-carboline alkaloids have been isolated from natural sources to date, 1 is the first ß-carboline derivative containing a sulfate group. Compound 2 inhibited chymotrypsin-like activity of the proteasome and Ubc13 (E2)-Uev1A interaction with IC50 values of 4 and 5 µg/mL, respectively, whereas 1 had little effect on the activity or interaction.
Subject(s)
Alkaloids/isolation & purification , Carbolines/isolation & purification , Porifera/chemistry , Proteasome Inhibitors/pharmacology , Alkaloids/pharmacology , Animals , Carbolines/pharmacology , Humans , Liver/drug effects , Liver/enzymology , Molecular Structure , Proteasome Inhibitors/isolation & purification , Rats , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
USP7, a deubiquitylating enzyme hydrolyzing the isopeptide bond at the C-terminus of ubiquitin, is an emerging cancer target. We isolated spongiacidin C from the marine sponge Stylissa massa as the first USP7 inhibitor from a natural source. This compound inhibited USP7 most strongly with an IC50 of 3.8 µM among several USP family members tested.
Subject(s)
Porifera/chemistry , Protease Inhibitors/pharmacology , Pyrroles/pharmacology , Ubiquitin Thiolesterase/antagonists & inhibitors , Animals , Dose-Response Relationship, Drug , Humans , Molecular Structure , Protease Inhibitors/chemistry , Pyrroles/chemistry , Pyrroles/isolation & purification , Structure-Activity Relationship , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Peptidase 7ABSTRACT
Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 µM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 µM.
Subject(s)
Propylene Glycols/isolation & purification , Propylene Glycols/pharmacology , Proto-Oncogene Proteins c-mdm2/drug effects , Tumor Suppressor Protein p53/drug effects , Urochordata/chemistry , Animals , Humans , Molecular Structure , Propanolamines , Propylene Glycols/chemistry , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolismABSTRACT
B-cell translocation gene 1 and 2 (BTG1 and BTG2) are members of the BTG/Tob antiproliferative protein family, which is able to regulate the cell cycle and cell proliferation. We previously reported that BTG1, BTG2, Tob, and Tob2 are degraded via the ubiquitin-proteasome pathway. In this study, we investigated the mechanism of polyubiquitination of BTG1 and BTG2. Since the Skp1-Cdc53/Cullin 1-F-box protein (SCF) complex functions as one of the major ubiquitin ligases for cell cycle regulation, we first examined interactions between BTG proteins and components of the SCF complex, and found that BTG1 and BTG2 were capable of interacting with the SCF complex containing Cullin-1 (a scaffold protein) and Skp1 (a linker protein). As the SCF complex can ubiquitinate various target proteins by substituting different F-box proteins as subunits that recognize different target proteins, we next examined which F-box proteins could bind the two BTG proteins, and found that Skp2, ß-transducin repeat-containing protein 1 (ßTrCP1), and ßTrCP2 were able to associate with both BTG1 and BTG2. Furthermore, we obtained evidence showing that ßTrCP1 enhanced the polyubiquitination of both BTG1 and BTG2 more efficiently than Skp2 did, and that an F-box truncated mutant of ßTrCP1 had a dominant negative effect on this polyubiquitination. Thus, we propose that BTG1 and BTG2 are subjected to polyubiquitination, more efficiently when it is mediated by SCFßTrCP than by SCFSkp2.
Subject(s)
B-Lymphocytes/metabolism , Cell Cycle , Immediate-Early Proteins/metabolism , Neoplasm Proteins/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitination , beta-Transducin Repeat-Containing Proteins/metabolism , Cell Cycle Checkpoints , HeLa Cells , Humans , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein SubunitsABSTRACT
Two new dimeric sterols, manadosterols A (1) and B (2), were isolated from the marine sponge Lissodendryx fibrosa collected in Indonesia. The two compounds are comprised of two sulfonated sterol cores connected through the respective side chains. Manadosterols A (1) and B (2) inhibited the Ubc13-Uev1A interaction with IC(50) values of 0.09 and 0.13 µM, respectively. They are the second and third natural compounds showing inhibitory activities against the Ubc13-Uev1A interaction and are more potent than leucettamol A (IC(50), 106 µM), the first such inhibitor, isolated from another marine sponge.
Subject(s)
Porifera/chemistry , Sterols/isolation & purification , Sterols/pharmacology , Animals , Drug Screening Assays, Antitumor , Indonesia , Inhibitory Concentration 50 , Marine Biology , Molecular Structure , Sphingolipids/pharmacology , Sterols/chemistryABSTRACT
Hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-ß-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Hyrtioreticulins A and B (1 and 2) inhibited ubiquitin-activating enzyme (E1) with IC(50) values of 0.75 and 11µg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A, largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent E1 inhibitor.
Subject(s)
Enzyme Inhibitors/chemistry , Indole Alkaloids/chemistry , Porifera/chemistry , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Animals , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/metabolism , Indole Alkaloids/isolation & purification , Indole Alkaloids/metabolism , Magnetic Resonance Spectroscopy , Molecular Conformation , Ubiquitin-Activating Enzymes/metabolismABSTRACT
P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹4-Cys³9 bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹4-Cys³9 bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.
Subject(s)
Cysteine/chemistry , Ovomucin/chemistry , Ovomucin/pharmacology , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/pharmacology , Animals , Binding Sites , Birds , Hydrogen-Ion Concentration , Hydrolysis , Models, Molecular , ThermodynamicsABSTRACT
Salsolinol (1), a tetrahydroisoquinoline alkaloid, was isolated from the marine sponge Xestospongia cf. vansoesti collected in Indonesia as a proteasome inhibitor, along with three salsolinol derivatives, norsalsolinol (2), cis-4-hydroxysalsolinol (3), and trans-4-hydroxysalsolinol (4). Compounds 1 and 2 inhibited the chymotrypsin-like activity of the proteasome with IC(50) values of 50 and 32 µg/ml, respectively, but 3 and 4 showed no inhibitory effect even at 100 µg/ml.
Subject(s)
Alkaloids/isolation & purification , Isoquinolines/isolation & purification , Proteasome Inhibitors , Tetrahydroisoquinolines/isolation & purification , Xestospongia/chemistry , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , HeLa Cells , Humans , Isoquinolines/chemistry , Isoquinolines/pharmacology , Proteasome Endopeptidase Complex/metabolism , Tetrahydroisoquinolines/chemistry , Tetrahydroisoquinolines/pharmacologyABSTRACT
BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.
Subject(s)
Molecular Chaperones/metabolism , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Animals , Genes, MHC Class I , HeLa Cells , Humans , Major Histocompatibility Complex , Mice , Molecular Chaperones/genetics , Molecular Sequence Data , NIH 3T3 Cells , Peptides/genetics , Polyubiquitin/metabolism , Protein Synthesis Inhibitors/metabolism , Puromycin , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tissue DistributionABSTRACT
Aaptamine (1), isoaaptamine (2), and demethylaaptamine (3) were isolated from the marine sponge Aaptossuberitoides collected in Indonesia as inhibitors of the proteasome. They inhibited the chymotrypsin-like and caspase-like activities of the proteasome with IC(50) values of 1.6-4.6 microg/mL, while they showed less inhibition of the trypsin-like activity of the proteasome. The three compounds showed cytotoxic activities against HeLa cells, but their cytotoxicity did not correlate with their potency as proteasome inhibitors, strongly suggesting that their proteasomal inhibitory activity is dispensable to their cytotoxicity.
Subject(s)
Naphthyridines/pharmacology , Porifera/chemistry , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Animals , HeLa Cells , Humans , Naphthyridines/isolation & purification , Protease Inhibitors/isolation & purificationABSTRACT
The ubiquitin-proteasome system plays a critical role in selective protein degradation and regulates almost all cellular events such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein quality control, development, and neuronal function. The recent approval of bortezomib, a synthetic proteasome inhibitor, for the treatment of relapsed multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and ubiquitinating and deubiquitinating enzymes as well as the delivery system. To date, various synthetic and natural products have been reported to inhibit the components of the ubiquitin-proteasome system. Here, we review natural products targeting the ubiquitin-proteasome system as well as synthetic compounds with potent inhibitory effects.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/drug effects , Ubiquitin/metabolism , Aldehydes/chemistry , Aldehydes/pharmacology , Boronic Acids/chemistry , Boronic Acids/pharmacology , Ketones/chemistry , Ketones/pharmacology , Lactones/chemistry , Lactones/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protease Inhibitors/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteasome InhibitorsABSTRACT
Several 9-(2-C-cyano-2-deoxy-l-beta-D-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (2, CNDAG) as well as the 2-amino-6-substituted-purine derivatives 3, 4 and 5 inhibited KSHV-positive cell growth but showed no cytotoxicity against KSHV-negative cells at >15 muM concentrations. Therefore, it was found that compounds 2, 3, 4 and 5 showed selective cytotoxicity against PEL cells infected with KSHV.
Subject(s)
Antimetabolites, Antineoplastic/chemistry , Arabinonucleosides/chemistry , Guanosine/analogs & derivatives , Herpesvirus 8, Human , Lymphoma, B-Cell/drug therapy , Sarcoma, Kaposi/drug therapy , Antimetabolites, Antineoplastic/chemical synthesis , Antimetabolites, Antineoplastic/therapeutic use , Arabinonucleosides/chemical synthesis , Arabinonucleosides/therapeutic use , Cell Line , Cell Line, Transformed , Guanosine/chemical synthesis , Guanosine/chemistry , Guanosine/therapeutic use , HumansABSTRACT
The development of potent proteasome inhibitors based on the stereochemical diversity-oriented strategy using a conformationally rigid cyclopropane structure was investigated. Thus, a series of stereo- and regioisomeric analogs of belactosin A (2), a cyclopropane amino acid (methanoamino acid)-containing tripeptidic proteasome inhibitor, were designed, in which the central cyclopropane amino acid part was replaced with the corresponding stereo- or regioisomer. Using a series of stereoisomeric cyclopropane amino acid equivalents with the cis/trans, D/L, and syn/anti stereochemical diversity, which were previously developed by us, as key units, the target compounds were successfully synthesized. Biological evaluation showed that, as expected, compound activity changed depending on the stereochemistry of the central cyclopropane amino acid part: the trans/L-syn-isomer 7 and the cis/L-anti-isomer 9 were more than twice as potent as natural belactosin A (trans/L-anti-isomer). Furthermore, the tripeptidic compound 13, the synthetic precursor for the unnatural cis/L-anti-isomer 9, was identified as a highly potent proteasome inhibitor. This compound, which is 20 times as potent as belactosin A and is even more potent than the well-known inhibitor lactacystin (4), may be an effective lead for developing clinically useful anticancer drugs. These results show that the stereochemical diversity-oriented approach can be a powerful strategy for the development of highly active compounds in medicinal chemical studies.