1,003 results on '"Goldberg, A."'
Search Results
2. Loss of myeloid lipoprotein lipase exacerbates adipose tissue fibrosis with collagen VI deposition and hyperlipidemia in leptin-deficient obese mice
- Author
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Takahashi, Manabu, primary, Yamamuro, Daisuke, additional, Wakabayashi, Tetsuji, additional, Takei, Akihito, additional, Takei, Shoko, additional, Nagashima, Shuichi, additional, Okazaki, Hiroaki, additional, Ebihara, Ken, additional, Yagyu, Hiroaki, additional, Takayanagi, Yuki, additional, Onaka, Tatsushi, additional, Goldberg, Ira J., additional, and Ishibashi, Shun, additional
- Published
- 2022
- Full Text
- View/download PDF
3. The nepenthesin insert in the Plasmodium falciparum aspartic protease plasmepsin V is necessary for enzyme function
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Polino, Alexander J., primary, Miller, Justin J., additional, Bhakat, Soumendranath, additional, Mukherjee, Sumit, additional, Bobba, Suhas, additional, Bowman, Gregory R., additional, and Goldberg, Daniel E., additional
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- 2022
- Full Text
- View/download PDF
4. Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation
- Author
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Collins, Galen Andrew, primary, Sha, Zhe, additional, Kuo, Chueh-Ling, additional, Erbil, Beyza, additional, and Goldberg, Alfred L., additional
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- 2022
- Full Text
- View/download PDF
5. Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites
- Author
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Frasse, Philip M., primary, Miller, Justin J., additional, Polino, Alexander J., additional, Soleimani, Ebrahim, additional, Zhu, Jian-She, additional, Jakeman, David L., additional, Jez, Joseph M., additional, Goldberg, Daniel E., additional, and Odom John, Audrey R., additional
- Published
- 2022
- Full Text
- View/download PDF
6. Malaria parasite plasmepsins: More than just plain old degradative pepsins
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Eva S. Istvan, Daniel E. Goldberg, Armiyaw S. Nasamu, and Alexander J. Polino
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0301 basic medicine ,Plasmodium ,Aspartic Acid Proteases ,medicine.medical_treatment ,Protozoan Proteins ,Plasmepsin ,Vacuole ,Biochemistry ,Antimalarials ,03 medical and health sciences ,Organelle ,medicine ,Aspartic Acid Endopeptidases ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Organism ,Genetics ,Nepenthesin ,Protease ,030102 biochemistry & molecular biology ,biology ,Effector ,JBC Reviews ,Cell Biology ,biology.organism_classification ,Malaria ,030104 developmental biology - Abstract
Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium. Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism.
- Published
- 2020
7. Substrate recognition by the Pseudomonas aeruginosa EF-Tu–modifying methyltransferase EftM
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Joanna B. Goldberg, Debayan Dey, Samantha M. Prezioso, Emily G. Kuiper, Paige A. LaMore, Joshua P. Owings, and Graeme L. Conn
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0301 basic medicine ,chemistry.chemical_classification ,Rossmann fold ,Methyltransferase ,030102 biochemistry & molecular biology ,Chemistry ,Pseudomonas aeruginosa ,Mutagenesis (molecular biology technique) ,Peptide ,Cell Biology ,medicine.disease_cause ,Biochemistry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Protein methylation ,medicine ,Homology modeling ,Molecular Biology ,EF-Tu - Abstract
The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface. We also show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5–6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminal sequence to facilitate Lys-5 modification. Our findings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.
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- 2019
8. Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane
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Henning Urlaub, Marret Müller, Christof Lenz, Ralph H. Kehlenbach, Martin W. Goldberg, and Christina James
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Proteomics ,0301 basic medicine ,Nuclear Envelope ,Immunoelectron microscopy ,Vesicular Transport Proteins ,Emerin ,Endoplasmic Reticulum ,Biochemistry ,03 medical and health sciences ,Stable isotope labeling by amino acids in cell culture ,Protein Interaction Mapping ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Humans ,Inner membrane ,Protein Interaction Maps ,Nuclear pore ,Microscopy, Immunoelectron ,Molecular Biology ,Sirolimus ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,Endoplasmic reticulum ,Membrane Proteins ,Nuclear Proteins ,Cell Biology ,VAPB ,Endonucleases ,Multifunctional Enzymes ,Amino acid ,Cell biology ,DNA-Binding Proteins ,Protein Transport ,030104 developmental biology ,Isotope Labeling ,HeLa Cells ,Protein Binding ,Transcription Factors - Abstract
Vesicle-associated membrane protein–associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.
- Published
- 2019
9. The nepenthesin insert in the Plasmodium falciparum aspartic protease plasmepsin V is necessary for enzyme function
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Alexander J. Polino, Justin J. Miller, Soumendranath Bhakat, Sumit Mukherjee, Suhas Bobba, Gregory R. Bowman, and Daniel E. Goldberg
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Plasmodium falciparum ,Protozoan Proteins ,Aspartic Acid Endopeptidases ,Humans ,Disulfides ,Cell Biology ,Malaria, Falciparum ,Molecular Biology ,Biochemistry ,Pepsin A - Abstract
Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malarial parasite Plasmodium falciparum. Previous work has shown PM V to be an endoplasmic reticulum-resident protease that processes parasite proteins destined for export into the host cell. Depletion or inhibition of the enzyme is lethal during asexual replication within red blood cells as well as during the formation of sexual stage gametocytes. The structure of the Plasmodium vivax PM V has been characterized by X-ray crystallography, revealing a canonical pepsin fold punctuated by structural features uncommon to secretory aspartic proteases; however, the function of this unique structure is unclear. Here, we used parasite genetics to probe these structural features by attempting to rescue lethal PM V depletion with various mutant enzymes. We found an unusual nepenthesin 1-type insert in the PM V gene to be essential for parasite growth and PM V activity. Mutagenesis of the nepenthesin insert suggests that both its amino acid sequence and one of the two disulfide bonds that undergird its structure are required for the insert's role in PM V function. Furthermore, molecular dynamics simulations paired with Markov state modeling suggest that mutations to the nepenthesin insert may allosterically affect PM V catalysis through multiple mechanisms. Taken together, these data provide further insights into the structure of the P. falciparum PM V protease.
- Published
- 2022
10. Loss of myeloid lipoprotein lipase exacerbates adipose tissue fibrosis with collagen VI deposition and hyperlipidemia in leptin-deficient obese mice
- Author
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Manabu Takahashi, Daisuke Yamamuro, Tetsuji Wakabayashi, Akihito Takei, Shoko Takei, Shuichi Nagashima, Hiroaki Okazaki, Ken Ebihara, Hiroaki Yagyu, Yuki Takayanagi, Tatsushi Onaka, Ira J. Goldberg, and Shun Ishibashi
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Collagen Type IV ,Hypertriglyceridemia ,Leptin ,Adipose Tissue, White ,Lipoproteins ,Mice, Obese ,Cell Biology ,Fibrosis ,Biochemistry ,Actins ,Lipoprotein Lipase ,Mice ,Animals ,Obesity ,Molecular Biology ,Triglycerides - Abstract
During obesity, tissue macrophages increase in number and become proinflammatory, thereby contributing to metabolic dysfunction. Lipoprotein lipase (LPL), which hydrolyzes triglyceride in lipoproteins, is secreted by macrophages. However, the role of macrophage-derived LPL in adipose tissue remodeling and lipoprotein metabolism is largely unknown. To clarify these issues, we crossed leptin-deficient Lep
- Published
- 2022
11. Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation
- Author
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Galen Andrew Collins, Zhe Sha, Chueh-Ling Kuo, Beyza Erbil, and Alfred L. Goldberg
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Mammals ,Proteasome Endopeptidase Complex ,Nelfinavir ,Ubiquitin ,Proteolysis ,Animals ,Cell Biology ,Proteasome Inhibitors ,Molecular Biology ,Biochemistry - Abstract
Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors.
- Published
- 2022
12. Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites
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Philip M. Frasse, Justin J. Miller, Alexander J. Polino, Ebrahim Soleimani, Jian-She Zhu, David L. Jakeman, Joseph M. Jez, Daniel E. Goldberg, and Audrey R. Odom John
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0303 health sciences ,Erythrocytes ,Glycosylphosphatidylinositols ,030306 microbiology ,Plasmodium falciparum ,Protozoan Proteins ,Cell Biology ,Biochemistry ,03 medical and health sciences ,Phosphotransferases (Phosphomutases) ,Animals ,Humans ,Malaria, Falciparum ,Molecular Biology ,030304 developmental biology - Abstract
The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.
- Published
- 2022
13. Malaria parasite plasmepsins: More than just plain old degradative pepsins
- Author
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Nasamu, Armiyaw S., primary, Polino, Alexander J., additional, Istvan, Eva S., additional, and Goldberg, Daniel E., additional
- Published
- 2020
- Full Text
- View/download PDF
14. Substrate recognition by the Pseudomonas aeruginosa EF-Tu–modifying methyltransferase EftM
- Author
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Kuiper, Emily G., primary, Dey, Debayan, additional, LaMore, Paige A., additional, Owings, Joshua P., additional, Prezioso, Samantha M., additional, Goldberg, Joanna B., additional, and Conn, Graeme L., additional
- Published
- 2019
- Full Text
- View/download PDF
15. Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane
- Author
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James, Christina, primary, Müller, Marret, additional, Goldberg, Martin W., additional, Lenz, Christof, additional, Urlaub, Henning, additional, and Kehlenbach, Ralph H., additional
- Published
- 2019
- Full Text
- View/download PDF
16. Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies
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Sha, Zhe, primary, Blyszcz, Tamara, additional, González-Prieto, Román, additional, Vertegaal, Alfred C.O., additional, and Goldberg, Alfred L., additional
- Published
- 2019
- Full Text
- View/download PDF
17. The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis
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Hyoung Tae Kim and Alfred L. Goldberg
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0301 basic medicine ,Proteasome Endopeptidase Complex ,Proteolysis ,ATPase ,Allosteric regulation ,Protein degradation ,Biochemistry ,Deubiquitinating enzyme ,Mice ,03 medical and health sciences ,Adenosine Triphosphate ,Allosteric Regulation ,Ubiquitin ,ATP hydrolysis ,medicine ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,Mice, Knockout ,medicine.diagnostic_test ,biology ,Ubiquitination ,Cell Biology ,Fibroblasts ,Embryo, Mammalian ,Ubiquitinated Proteins ,Cell biology ,030104 developmental biology ,Proteasome ,Protein Synthesis and Degradation ,Mutation ,Trans-Activators ,biology.protein ,Ubiquitin Thiolesterase - Abstract
The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5′-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30–40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.
- Published
- 2017
18. Identification of a Substrate-selective Exosite within the Metalloproteinase Anthrax Lethal Factor
- Author
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Benjamin E. Turk, Allison B. Goldberg, Hua Jane Lou, Eunice Cho, and Chad J. Miller
- Subjects
0301 basic medicine ,MAP Kinase Signaling System ,Anthrax toxin ,medicine.medical_treatment ,Bacterial Toxins ,Mutation, Missense ,Mitogen-activated protein kinase kinase ,Biology ,Biochemistry ,MKKS ,Mice ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Mitogen-Activated Protein Kinase Kinases ,Antigens, Bacterial ,Metalloproteinase ,Protease ,030102 biochemistry & molecular biology ,Kinase ,Cell Biology ,030104 developmental biology ,Amino Acid Substitution ,Bacillus anthracis ,Enzymology ,Signal transduction ,Apoptosis Regulatory Proteins - Abstract
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.
- Published
- 2017
19. Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis
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Alexander Wlodawer, Mi Li, Poorva Dharkar, Alfred L. Goldberg, Olga Kandror, Tatos Akopian, and Michael R. Maurizi
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0301 basic medicine ,Stereochemistry ,medicine.medical_treatment ,ATPase ,030106 microbiology ,Allosteric regulation ,Plasma protein binding ,Biology ,Protein degradation ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Allosteric Regulation ,Bacterial Proteins ,Multienzyme Complexes ,medicine ,Binding site ,Protein Structure, Quaternary ,Molecular Biology ,Binding Sites ,Protease ,Dipeptide ,Serine Endopeptidases ,Dipeptides ,Mycobacterium tuberculosis ,Cell Biology ,030104 developmental biology ,chemistry ,Enzymology ,biology.protein ,Oxyanion hole ,Protein Binding - Abstract
The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.
- Published
- 2016
20. The N-terminal Set-β Protein Isoform Induces Neuronal Death
- Author
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Michael P. Vitek, Yan Wang, Melina I. Morkin, Stephanie G. Fernandez, Gregory M. Mlacker, Dmitry Velmeshev, Peter X. Shaw, Alan Sang, Jeffrey L. Goldberg, Susan M. Dombrowski, Xiongfei Liu, Karan H. Patel, Han Gao, and Ephraim F. Trakhtenberg
- Subjects
Retinal Ganglion Cells ,Gene isoform ,Blotting, Western ,Fluorescent Antibody Technique ,Apoptosis ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,Immunoenzyme Techniques ,Rats, Sprague-Dawley ,Mice ,Neurobiology ,medicine ,Animals ,Protein Isoforms ,Histone Chaperones ,RNA, Messenger ,Nuclear protein ,Molecular Biology ,Cells, Cultured ,Cell Proliferation ,Neurons ,Oncogene Proteins ,Reverse Transcriptase Polymerase Chain Reaction ,fungi ,Alternative splicing ,food and beverages ,Nuclear Proteins ,Cell Biology ,Subcellular localization ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,Mice, Inbred C57BL ,Alternative Splicing ,medicine.anatomical_structure ,nervous system ,Animals, Newborn ,Retinal ganglion cell ,Cytoplasm ,Neuron ,Carrier Proteins ,Nuclear localization sequence - Abstract
Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death.
- Published
- 2015
21. The Heme Biosynthesis Pathway Is Essential for Plasmodium falciparum Development in Mosquito Stage but Not in Blood Stages
- Author
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Kazutoyo Miura, Paul A. Sigala, Hangjun Ke, Carole A. Long, Michael W. Mather, Joanne M. Morrisey, Daniel E. Goldberg, Jeffrey P. Henderson, Akhil B. Vaidya, and Jan R. Crowley
- Subjects
Male ,Erythrocytes ,Hemeprotein ,Plasmodium falciparum ,Heme ,Microbiology ,Biochemistry ,Cofactor ,Gene Knockout Techniques ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Anopheles ,parasitic diseases ,Animals ,Humans ,Molecular Biology ,Anopheles stephensi ,biology ,Hemozoin ,Cell Biology ,Ferrochelatase ,biology.organism_classification ,chemistry ,biology.protein ,Female ,5-Aminolevulinate Synthetase - Abstract
Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
- Published
- 2014
22. Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins
- Author
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Leigh A. Baxt, Gavin MacBeath, Marcia B. Goldberg, Jason E. Heindl, Brian C. Russo, John E. Allen, Emily Rose Kahoud, Chae-ryun Yi, Soo Young Lee, and Bobby Brooke Herrera
- Subjects
Amino Acid Motifs ,Molecular Sequence Data ,PDZ domain ,Intracellular Space ,Protein Array Analysis ,Saccharomyces cerevisiae ,Microbiology ,Biochemistry ,Protein–protein interaction ,Shigella flexneri ,Bacterial Proteins ,Humans ,Protein Interaction Domains and Motifs ,Secretion ,Amino Acid Sequence ,Molecular Biology ,Conserved Sequence ,Protein Kinase C ,Protein kinase C ,Adaptor Proteins, Signal Transducing ,LIM domain ,Focal Adhesions ,biology ,Effector ,Cell Biology ,LIM Domain Proteins ,biology.organism_classification ,Cell biology ,Cytoskeletal Proteins ,HEK293 Cells ,Mutant Proteins ,sense organs ,Shigella ,Peptides ,Postsynaptic density ,HeLa Cells ,Protein Binding ,Signal Transduction - Abstract
Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.
- Published
- 2014
23. Kinase-associated Endopeptidase 1 (Kae1) Participates in an Atypical Ribosome-associated Complex in the Apicoplast of Plasmodium falciparum
- Author
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Anna Oksman, Jeremy P. Mallari, Barbara A. Vaupel, and Daniel E. Goldberg
- Subjects
TRNA modification ,Protein family ,Immunoprecipitation ,Molecular Sequence Data ,Plasmodium falciparum ,Protozoan Proteins ,Apicoplasts ,Biochemistry ,Ribosome ,Mass Spectrometry ,Conserved sequence ,Cytosol ,Nucleic Acids ,Animals ,Cluster Analysis ,Humans ,Parasites ,Amino Acid Sequence ,Protein Interaction Maps ,Molecular Biology ,Alleles ,Conserved Sequence ,Phylogeny ,Life Cycle Stages ,Apicoplast ,biology ,Molecular Sequence Annotation ,Cell Biology ,DNA, Protozoan ,biology.organism_classification ,Recombinant Proteins ,Cell Compartmentation ,Cell biology ,Transport protein ,Protein Transport ,RNA, Ribosomal ,Ribosomes ,Sequence Alignment ,Protein Binding - Abstract
The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in N(6)-threonylcarbamoyl adenosine tRNA modification, transcriptional regulation, and telomere homeostasis. These functions are performed in complex with a conserved core of protein binding partners. Herein we describe the localization, essentiality, and protein-protein interactions for Kae1 in the human malaria parasite Plasmodium falciparum. We found that the parasite expresses one Kae1 protein in the cytoplasm and a second protein in the apicoplast, a chloroplast remnant organelle involved in fatty acid, heme, and isoprenoid biosynthesis. To analyze the protein interaction networks for both Kae1 pathways, we developed a new proteomic cross-validation approach. This strategy compares immunoprecipitation-MS data sets across different cellular compartments to enrich for biologically relevant protein interactions. Our results show that cytoplasmic Kae1 forms a complex with Bud32 and Cgi121 as in other organisms, whereas apicoplast Kae1 makes novel interactions with multiple proteins in the apicoplast. Quantitative RT-PCR and immunoprecipitation studies indicate that apicoplast Kae1 and its partners interact specifically with the apicoplast ribosomes and with proteins involved in ribosome function. Together, these data indicate an expanded, apicoplast-specific role for Kae1 in the parasite.
- Published
- 2014
24. Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking
- Author
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Aileen Ren, Nicole Dudley-Rucker, Katrina A. Brandis, Hui Jiang, Anna Goldberg, Rohini Sidhu, Sarah Jinn, Daniel S. Ory, Jean E. Schaffer, Sarah E. Gale, and Stephen J. Langmade
- Subjects
health care facilities, manpower, and services ,education ,Molecular Sequence Data ,Mutant ,Intracellular Space ,CHO Cells ,Haploinsufficiency ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Mice ,chemistry.chemical_compound ,Cricetulus ,Ribonucleoproteins, Small Nucleolar ,Amphotericin B ,Cricetinae ,Animals ,Humans ,RNA, Small Nucleolar ,Small nucleolar RNA ,Molecular Biology ,health care economics and organizations ,Regulation of gene expression ,Base Sequence ,Endoplasmic reticulum ,Chinese hamster ovary cell ,Cell Membrane ,Biological Transport ,Cell Biology ,Methylation ,Non-coding RNA ,Lipids ,Molecular biology ,Rats ,stomatognathic diseases ,Cholesterol ,Gene Expression Regulation ,chemistry ,Genetic Loci ,Gene Knockdown Techniques ,Mutation ,Phosphatidylcholines ,Cholesteryl ester ,lipids (amino acids, peptides, and proteins) - Abstract
Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.
- Published
- 2013
25. SIRT1 Protein, by Blocking the Activities of Transcription Factors FoxO1 and FoxO3, Inhibits Muscle Atrophy and Promotes Muscle Growth
- Author
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Alfred L. Goldberg and Dong-Hoon Lee
- Subjects
Male ,medicine.medical_specialty ,Cachexia ,animal structures ,Ubiquitin-Protein Ligases ,Muscle Proteins ,Biology ,Biochemistry ,Muscle hypertrophy ,Tripartite Motif Proteins ,Mice ,Phosphatidylinositol 3-Kinases ,Atrophy ,Sirtuin 1 ,Internal medicine ,medicine ,Animals ,Muscle, Skeletal ,Molecular Biology ,Denervation ,SKP Cullin F-Box Protein Ligases ,Forkhead Box Protein O1 ,fungi ,Forkhead Box Protein O3 ,Skeletal muscle ,Forkhead Transcription Factors ,Fasting ,Cell Biology ,medicine.disease ,Muscle atrophy ,Muscular Atrophy ,enzymes and coenzymes (carbohydrates) ,Endocrinology ,medicine.anatomical_structure ,FOXO3 ,lipids (amino acids, peptides, and proteins) ,biological phenomena, cell phenomena, and immunity ,medicine.symptom ,ITGA7 ,human activities ,Proto-Oncogene Proteins c-akt ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction ,Deacetylase activity - Abstract
In several cell types, the protein deacetylase SIRT1 regulates the activities of FoxO transcription factors whose activation is critical in muscle atrophy. However, the possible effects of SIRT1 on the activity of FoxOs in skeletal muscle and on the regulation of muscle size have not been investigated. Here, we show that after food deprivation, SIRT1 levels fall dramatically in type II skeletal muscles (tibialis anterior), which show marked atrophy, unlike in the liver (where SIRT1 rises) or heart or the soleus, a type I muscle (where SIRT1 is unchanged). Maintenance of high SIRT1 levels by electroporation in mouse muscle inhibits markedly the muscle wasting induced by fasting as well as by denervation, and these protective effects require its deacetylase activity. SIRT1 overexpression reduces muscle wasting by blocking the activation of FoxO1 and 3. It thus prevents the induction of key atrogenes, including the muscle-specific ubiquitin ligases, atrogin1 and MuRF1, and multiple autophagy (Atg) genes and the increase in overall proteolysis. In normal muscle, SIRT1 overexpression by electroporation causes rapid fiber hypertrophy without, surprisingly, activation of the PI3K-AKT signaling pathway. Thus, SIRT1 activation favors postnatal muscle growth, and its fall appears to be critical for atrophy during fasting. Consequently, SIRT1 activation represents an attractive possible pharmacological approach to prevent muscle wasting and cachexia.
- Published
- 2013
26. The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis
- Author
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Weinhäupl, Katharina, primary, Brennich, Martha, additional, Kazmaier, Uli, additional, Lelievre, Joel, additional, Ballell, Lluis, additional, Goldberg, Alfred, additional, Schanda, Paul, additional, and Fraga, Hugo, additional
- Published
- 2018
- Full Text
- View/download PDF
27. Adipose-specific Lipoprotein Lipase Deficiency More Profoundly Affects Brown than White Fat Biology
- Author
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Sheila M. O'Byrne, William S. Blaner, Manabu Takahashi, Konstantinos Drosatos, Yunying Hu, Ni Huiping Son, Ira J. Goldberg, Itsaso Garcia-Arcos, Yaeko Hiyama, Chuchun L. Chang, Richard J. Deckelbaum, Joseph C. Obunike, Kalyani G. Bharadwaj, Marit Westerterp, Hongfeng Jiang, Hiroaki Yagyu, Center for Liver, Digestive and Metabolic Diseases (CLDM), Translational Immunology Groningen (TRIGR), and Medical Biochemistry
- Subjects
Adipose Tissue/metabolism ,Male ,Adipose Tissue, Brown/metabolism ,Adipose tissue ,White adipose tissue ,Biochemistry ,Mice ,chemistry.chemical_compound ,Adipose Tissue, Brown ,Adipocyte ,Chylomicrons ,Brown adipose tissue ,Adipocytes ,Bone Marrow Transplantation ,Mice, Knockout ,Lipoprotein lipase ,Adipocytes/cytology ,digestive, oral, and skin physiology ,Lipids ,White/metabolism ,Triglycerides/blood ,Phenotype ,medicine.anatomical_structure ,Adipose Tissue ,lipids (amino acids, peptides, and proteins) ,medicine.medical_specialty ,Chylomicrons/pharmacokinetics ,Adipose Tissue, White/metabolism ,Knockout ,Adipose Tissue, White ,Lipolysis ,Lipids/chemistry ,Biology ,Brown/metabolism ,Internal medicine ,medicine ,Animals ,Molecular Biology ,Triglycerides ,Triglyceride ,Macrophages ,Macrophages/cytology ,nutritional and metabolic diseases ,Cell Biology ,Lipoprotein Lipase/deficiency ,Lipoprotein Lipase ,Endocrinology ,chemistry ,Chylomicron - Abstract
Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the "gatekeeper" for tissue lipid distribution.
- Published
- 2013
28. Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells
- Author
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Susan E. Quaggin, Ling Xia, I. George Fantus, Howard J. Goldberg, Anu Shah, and Ken W. Lee
- Subjects
inorganic chemicals ,Collagen Type IV ,Mitochondrial ROS ,Thioredoxin-Interacting Protein ,Thioredoxin reductase ,Blotting, Western ,Gene Expression ,medicine.disease_cause ,Biochemistry ,Mice ,Thioredoxins ,medicine ,Animals ,Molecular Biology ,Cells, Cultured ,Mice, Knockout ,Mice, Inbred C3H ,Microscopy, Confocal ,NADPH oxidase ,biology ,Mesangial cell ,Reverse Transcriptase Polymerase Chain Reaction ,NADPH Oxidases ,NOX4 ,Cell Biology ,Molecular biology ,Mitochondria ,Metabolism ,Glucose ,NADPH Oxidase 4 ,Mesangial Cells ,biology.protein ,RNA Interference ,Carrier Proteins ,Reactive Oxygen Species ,TXNIP ,Oxidative stress ,Signal Transduction - Abstract
Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.
- Published
- 2013
29. A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase
- Author
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Ayala Gold, Gideon Coster, Darlene Chen, David G. Schatz, and Michal Goldberg
- Subjects
Repetitive Sequences, Amino Acid ,DNA damage ,DNA repair ,Immunology ,Amino Acid Motifs ,Cell Cycle Proteins ,chemical and pharmacologic phenomena ,Models, Biological ,Peptide Mapping ,Biochemistry ,Histones ,Cell Line, Tumor ,Recombinase ,Humans ,Phosphorylation ,Binding site ,VDJ Recombinases ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Homeodomain Proteins ,biology ,BRCA1 Protein ,V(D)J recombination ,Histone H2AX ,Nuclear Proteins ,hemic and immune systems ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,MDC1 ,Histone ,Trans-Activators ,biology.protein - Abstract
The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.
- Published
- 2012
30. Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes
- Author
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Prasanna Venkatraman, Rosanna Piccirillo, Raphael Hourez, Nidhi Bhutani, and Alfred L. Goldberg
- Subjects
Proteases ,Huntingtin ,Cathepsin L ,medicine.medical_treatment ,macromolecular substances ,Biology ,Biochemistry ,Mice ,medicine ,Animals ,Humans ,Muscle, Skeletal ,Molecular Biology ,Cathepsin ,Protease ,musculoskeletal, neural, and ocular physiology ,Neurodegeneration ,Neurodegenerative Diseases ,Cell Biology ,medicine.disease ,Cell biology ,Cytosol ,HEK293 Cells ,nervous system ,Proteasome ,Protein Synthesis and Degradation ,Cathepsin Z ,NIH 3T3 Cells ,biology.protein ,Lysosomes ,Peptides ,HeLa Cells - Abstract
In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.
- Published
- 2012
31. Cardiomyocyte Specific Deficiency of Serine Palmitoyltransferase Subunit 2 Reduces Ceramide but Leads to Cardiac Dysfunction
- Author
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Cheolsoo Choi, Yunying Hu, Tae-Sik Park, Ira J. Goldberg, Raffay S. Khan, Su-Yeon Lee, Yong-Moon Lee, Woo-Jin Park, In Sun Park, Mercy M. Davidson, Su-Jung Kim, Jung Ran Kim, Xian-Cheng Jiang, Kalyani G. Bharadwaj, and Kyong-Oh Shin
- Subjects
Blood Glucose ,Ceramide ,Blotting, Western ,Serine C-Palmitoyltransferase ,Biology ,Ceramides ,Biochemistry ,Mice ,chemistry.chemical_compound ,In Situ Nick-End Labeling ,Animals ,Molecular Biology ,Cells, Cultured ,Diacylglycerol kinase ,Mice, Knockout ,chemistry.chemical_classification ,Fatty acid metabolism ,Myocardium ,Serine C-palmitoyltransferase ,Fatty acid ,Heart ,Cell Biology ,Lipid signaling ,Lipids ,Sphingolipid ,Cell biology ,Mice, Inbred C57BL ,chemistry ,cardiovascular system ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin - Abstract
The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.
- Published
- 2012
32. Blm10 Protein Promotes Proteasomal Substrate Turnover by an Active Gating Mechanism
- Author
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Philipp C. Rommel, Daniel Finley, Tahel Noy, Lukas Jurzitza, Marion Schmidt, David M. Smith, Anne Legendre, Thomas Dange, Radames J. B. Cordero, and Alfred L. Goldberg
- Subjects
Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Activator (genetics) ,Saccharomyces cerevisiae ,Cell Biology ,Plasma protein binding ,Gating ,Biology ,Protein degradation ,Biochemistry ,Protein–protein interaction ,Cell biology ,Phenotype ,Proteasome ,Structural biology ,Protein Synthesis and Degradation ,Tyrosine ,Molecular Biology ,Protein Binding - Abstract
For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.
- Published
- 2011
33. Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts
- Author
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Jiyun Kwon, Benjamin D. Wheal, Ryan P.P. Shugg, Stephen M. Sims, S. Jeffrey Dixon, Natsuko Tanabe, Hong H. Chen, and Harvey A. Goldberg
- Subjects
musculoskeletal diseases ,integumentary system ,biology ,NFAT ,Cell Biology ,NFATC Transcription Factors ,Biochemistry ,Cell biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,stomatognathic system ,BAPTA ,chemistry ,Osteoclast ,Cancer research ,biology.protein ,medicine ,Osteopontin ,Molecular Biology ,Transcription factor ,Intracellular ,Integrin binding - Abstract
Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca2+-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca2+ (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) suppressed Ca2+ oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca2+-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.
- Published
- 2011
34. Inhibition of c-Jun-N-terminal Kinase Increases Cardiac Peroxisome Proliferator-activated Receptor α Expression and Fatty Acid Oxidation and Prevents Lipopolysaccharide-induced Heart Dysfunction
- Author
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Raffay S. Khan, P. Christian Schulze, Shunichi Homma, Konstantinos Drosatos, Ira J. Goldberg, Vassilis I. Zannis, and Zoi Drosatos-Tampakaki
- Subjects
Lipopolysaccharides ,medicine.medical_specialty ,Heart Diseases ,Lipopolysaccharide ,Muscle Proteins ,Peroxisome proliferator-activated receptor ,Inflammation ,Biology ,Biochemistry ,Cell Line ,Sepsis ,Mice ,chemistry.chemical_compound ,Internal medicine ,medicine ,Animals ,Myocytes, Cardiac ,PPAR alpha ,Receptor ,Molecular Biology ,Beta oxidation ,Anthracenes ,Mice, Knockout ,chemistry.chemical_classification ,Myocardium ,Fatty Acids ,c-jun ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,Peroxisome ,medicine.disease ,Metabolism ,Endocrinology ,Gene Expression Regulation ,chemistry ,medicine.symptom ,Oxidation-Reduction - Abstract
Septic shock results from bacterial infection and is associated with multi-organ failure, high mortality, and cardiac dysfunction. Sepsis causes both myocardial inflammation and energy depletion. We hypothesized that reduced cardiac energy production is a primary cause of ventricular dysfunction in sepsis. The JNK pathway is activated in sepsis and has also been implicated in impaired fatty acid oxidation in several tissues. Therefore, we tested whether JNK activation inhibits cardiac fatty acid oxidation and whether blocking JNK would restore fatty acid oxidation during LPS treatment. LPS treatment of C57BL/6 mice and adenovirus-mediated activation of the JNK pathway in cardiomyocytes inhibited peroxisome proliferator-activated receptor α expression and fatty acid oxidation. Surprisingly, none of the adaptive responses that have been described in other types of heart failure, such as increased glucose utilization, reduced αMHC:βMHC ratio or induction of certain microRNAs, occurred in LPS-treated mice. Treatment of C57BL/6 mice with a general JNK inhibitor (SP600125) increased fatty acid oxidation in mice and a cardiomyocyte-derived cell line. JNK inhibition also prevented LPS-mediated reduction in fatty acid oxidation and cardiac dysfunction. Inflammation was not alleviated in LPS-treated mice that received the JNK inhibitor. We conclude that activation of JNK signaling reduces fatty acid oxidation and prevents the peroxisome proliferator-activated receptor α down-regulation that occurs with LPS.
- Published
- 2011
35. Alternative Mitochondrial Electron Transfer as a Novel Strategy for Neuroprotection
- Author
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Yi Wen, Ethan Poteet, Shao-Hua Yang, Hua She, James W. Simpkins, Wenjun Li, Hai Qian, Zixu Mao, Xiaohua Ju, Luokun Xie, Ran Liu, Marian Marvin, Liang-Jun Yan, Cong Tan, and Matthew S. Goldberg
- Subjects
Male ,Substantia nigra ,Mitochondrion ,medicine.disease_cause ,Biochemistry ,Neuroprotection ,Cell Line ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Oxygen Consumption ,Neurobiology ,Rotenone ,medicine ,Animals ,Enzyme Inhibitors ,Parkinson Disease, Secondary ,Molecular Biology ,Neurons ,biology ,Uncoupling Agents ,Cytochrome c ,Neurodegeneration ,Cell Biology ,medicine.disease ,Mitochondria ,Rats ,Cell biology ,Methylene Blue ,Substantia Nigra ,Neuroprotective Agents ,Electron Transport Chain Complex Proteins ,chemistry ,Anaerobic glycolysis ,biology.protein ,Glycolysis ,Oxidative stress - Abstract
Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.
- Published
- 2011
36. Perlecan Mediates the Antiproliferative Effect of Apolipoprotein E on Smooth Muscle Cells
- Author
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Ira J. Goldberg, Sungshin Y. Choi, Joseph C. Obunike, Sivaram Pillarisetti, Itzhak D. Goldberg, Uday Saxena, and Latha Paka
- Subjects
Apolipoprotein E ,biology ,Cell growth ,Cell ,Cell Biology ,Heparan sulfate ,Perlecan ,Heparin ,Biochemistry ,Dermatan sulfate ,Cell biology ,carbohydrates (lipids) ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,medicine ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Thymidine ,Molecular Biology ,medicine.drug - Abstract
Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 μg/ml) increased 35SO4 incorporation into PG in media by 25–30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [3H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of35SO4 to [3H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.
- Published
- 1999
37. Unexpected Expression Pattern for Glycosylphosphatidylinositol-anchored HDL-binding Protein 1 (GPIHBP1) in Mouse Tissues Revealed by Positron Emission Tomography Scanning
- Author
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Michael M. Weinstein, Chika Nobumori, Koon-Pong Wong, Sung-Cheng Huang, Ira J. Goldberg, Tove Olafsen, Constance V. Voss, Vania E. Kenanova, André Bensadoun, Yiping Tu, Glen Young, Loren G. Fong, Anna M. Wu, Richard H. Barnes, Anne P. Beigneux, Stephen G. Young, and Brandon S.J. Davies
- Subjects
Endothelium ,Glycosylphosphatidylinositols ,Adipose tissue ,Mice, Transgenic ,Biology ,Models, Biological ,Biochemistry ,Mice ,medicine ,Animals ,Lipolysis ,Receptor ,Lung ,Molecular Biology ,Receptors, Lipoprotein ,Lipoprotein lipase ,Binding Sites ,Cell Membrane ,GPIHBP1 ,Skeletal muscle ,Cell Biology ,Lipids ,Molecular biology ,Capillaries ,Kinetics ,medicine.anatomical_structure ,Gene Expression Regulation ,Positron-Emission Tomography ,Immunology ,lipids (amino acids, peptides, and proteins) ,Lipoprotein - Abstract
Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), a GPI-anchored endothelial cell protein, binds lipoprotein lipase (LPL) and transports it into the lumen of capillaries where it hydrolyzes triglycerides in lipoproteins. GPIHBP1 is assumed to be expressed mainly within the heart, skeletal muscle, and adipose tissue, the sites where most lipolysis occurs, but the tissue pattern of GPIHBP1 expression has never been evaluated systematically. Because GPIHBP1 is found on the luminal face of capillaries, we predicted that it would be possible to define GPIHBP1 expression patterns with radiolabeled GPIHBP1-specific antibodies and positron emission tomography (PET) scanning. In Gpihbp1(-/-) mice, GPIHBP1-specific antibodies were cleared slowly from the blood, and PET imaging showed retention of the antibodies in the blood pools (heart and great vessels). In Gpihbp1(+/+) mice, the antibodies were cleared extremely rapidly from the blood and, to our surprise, were taken up mainly by lung and liver. Immunofluorescence microscopy confirmed the presence of GPIHBP1 in the capillary endothelium of both lung and liver. In most tissues with high levels of Gpihbp1 expression, Lpl expression was also high, but the lung was an exception (very high Gpihbp1 expression and extremely low Lpl expression). Despite low Lpl transcript levels, however, LPL protein was readily detectable in the lung, suggesting that some of that LPL originates elsewhere and then is captured by GPIHBP1 in the lung. In support of this concept, lung LPL levels were significantly lower in Gpihbp1(-/-) mice than in Gpihbp1(+/+) mice. In addition, Lpl(-/-) mice expressing human LPL exclusively in muscle contained high levels of human LPL in the lung.
- Published
- 2010
38. Chylomicron- and VLDL-derived Lipids Enter the Heart through Different Pathways
- Author
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Yunying Hu, Rajasekhar Ramakrishnan, William S. Blaner, Lesley Ann Huggins, Ira J. Goldberg, Yaeko Hiyama, Kalyani G. Bharadwaj, Nada A. Abumrad, and Gerald I. Shulman
- Subjects
chemistry.chemical_classification ,medicine.medical_specialty ,Lipoprotein lipase ,Very low-density lipoprotein ,Triglyceride ,biology ,CD36 ,digestive, oral, and skin physiology ,Fatty acid ,Blood lipids ,Cell Biology ,Biochemistry ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Internal medicine ,parasitic diseases ,medicine ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Chylomicron ,Lipoprotein - Abstract
Lipids circulate in the blood in association with plasma lipoproteins and enter the tissues either after hydrolysis or as non-hydrolyzable lipid esters. We studied cardiac lipids, lipoprotein lipid uptake, and gene expression in heart-specific lipoprotein lipase (LpL) knock-out (hLpL0), CD36 knock-out (Cd36−/−), and double knock-out (hLpL0/Cd36−/−-DKO) mice. Loss of either LpL or CD36 led to a significant reduction in heart total fatty acyl-CoA (control, 99.5 ± 3.8; hLpL0, 36.2 ± 3.5; Cd36−/−, 57.7 ± 5.5 nmol/g, p 10-fold and reduced HDL by >50%. After injection of these labeled chylomicrons in the different mice, chylomicron TG uptake was reduced by ∼70% and retinyl ester by ∼50% in hLpL0 hearts. Loss of CD36 did not alter either chylomicron TG or retinyl ester uptake. LpL loss did not affect uptake of remnant lipoproteins from ApoE knock-out mice. Our data are consistent with two pathways for fatty acid uptake; a CD36 process for VLDL-derived fatty acid and a non-CD36 process for chylomicron-derived fatty acid uptake. In addition, our data show that lipolysis is involved in uptake of core lipids from TG-rich lipoproteins.
- Published
- 2010
39. Identification of Dopamine D1–D3 Receptor Heteromers
- Author
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Oliver Saur, Rafael Franco, Steven R. Goldberg, Carmen Mazzola, Holger Stark, Chanel Barnes, Filippo Drago, Aroa Soriano, Bernard Le Foll, Daniel Marcellino, Kjell Fuxe, Antonio Cortés, Vicent Casadó, Sergi Ferré, and Carme Lluís
- Subjects
Agonist ,medicine.drug_class ,Cell Biology ,Pharmacology ,Biology ,Biochemistry ,Cell biology ,Dopamine receptor D1 ,Dopamine receptor D3 ,Dopamine ,Dopamine receptor D2 ,Dopamine receptor D5 ,medicine ,Enzyme-linked receptor ,Receptor ,Molecular Biology ,medicine.drug - Abstract
The function of dopamine D3 receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D1–D3 receptor heteromers and for an intramembrane D1–D3 receptor cross-talk in living cells and in the striatum. The formation of D1–D3 receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D1–D3 intramembrane receptor-receptor interaction was observed, by which D3 receptor stimulation enhances D1 receptor agonist affinity, indicating that the D1–D3 intramembrane receptor-receptor interaction is a biochemical characteristic of the D1–D3 receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D1 and D3 receptors. According to the synergistic D1–D3 intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D3 receptor stimulation potentiates D1 receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. The present study shows that a main functional significance of the D3 receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.
- Published
- 2008
40. The Molecular Basis of Retinoid Absorption
- Author
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Krzysztof Palczewski, Ellen Li, Thomas P. Johnston, Roseann Piantedosi, Nuttaporn Wongsiriroj, Ira J. Goldberg, and William S. Blaner
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medicine.medical_specialty ,medicine.drug_class ,Mutant ,Retinol ,Adipose tissue ,Cell Biology ,Biology ,Biochemistry ,In vitro ,chemistry.chemical_compound ,Endocrinology ,chemistry ,In vivo ,Internal medicine ,Acyltransferase ,medicine ,Retinoid ,Phosphatidylcholine—sterol O-acyltransferase ,Molecular Biology - Abstract
The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.
- Published
- 2008
41. Identification of a Novel Pool of Extracellular Pro-myostatin in Skeletal Muscle
- Author
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Sarah B. Anderson, Alfred L. Goldberg, and Malcolm Whitman
- Subjects
medicine.medical_specialty ,Xenopus ,Golgi Apparatus ,Smad2 Protein ,Myostatin ,Ligands ,Models, Biological ,Biochemistry ,Mice ,Transforming Growth Factor beta ,Internal medicine ,medicine ,Extracellular ,Animals ,Humans ,Muscle, Skeletal ,Molecular Biology ,Furin ,biology ,Skeletal muscle ,Cell Biology ,musculoskeletal system ,Proprotein convertase ,Cell biology ,medicine.anatomical_structure ,Endocrinology ,Gene Expression Regulation ,Latent TGF-beta Binding Proteins ,GDF11 ,biology.protein ,Proprotein Convertases ,Transforming growth factor - Abstract
Myostatin, a transforming growth factor-beta superfamily ligand, negatively regulates skeletal muscle growth. Generation of the mature signaling peptide requires cleavage of pro-myostatin by a proprotein convertase, which is thought to occur constitutively in the Golgi apparatus. In serum, mature myostatin is found in an inactive, non-covalent complex with its prodomain. We find that in skeletal muscle, unlike serum, myostatin is present extracellularly as uncleaved pro-myostatin. In cultured cells, co-expression of pro-myostatin and latent transforming growth factor-beta-binding protein-3 (LTBP-3) sequesters pro-myostatin in the extracellular matrix, and secreted pro-myostatin can be cleaved extracellularly by the proprotein convertase furin. Co-expression of LTBP-3 with myostatin reduces phosphorylation of Smad2, and ectopic expression of LTBP-3 in mature mouse skeletal muscle increases fiber area, consistent with reduction of myostatin activity. We propose that extracellular pro-myostatin constitutes the major pool of latent myostatin in muscle. Post-secretion activation of this pool by furin family proprotein convertases may therefore represent a major control point for activation of myostatin in skeletal muscle.
- Published
- 2008
42. The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis
- Author
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Kim, Hyoung Tae, primary and Goldberg, Alfred L., additional
- Published
- 2017
- Full Text
- View/download PDF
43. Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B
- Author
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Ren, Dapeng, primary, Fisher, Laura A., additional, Zhao, Jing, additional, Wang, Ling, additional, Williams, Byron C., additional, Goldberg, Michael L., additional, and Peng, Aimin, additional
- Published
- 2017
- Full Text
- View/download PDF
44. An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds
- Author
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Milstein, Michelle L., primary, Kimler, Victoria A., additional, Ghatak, Chiranjib, additional, Ladokhin, Alexey S., additional, and Goldberg, Andrew F.X., additional
- Published
- 2017
- Full Text
- View/download PDF
45. Regulation of ULK1 Expression and Autophagy by STAT1
- Author
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Goldberg, Alexander A., primary, Nkengfac, Bernard, additional, Sanchez, Anthony M.J., additional, Moroz, Nikolay, additional, Qureshi, Salman T., additional, Koromilas, Antonis E., additional, Wang, Shuo, additional, Burelle, Yan, additional, Hussain, Sabah N., additional, and Kristof, Arnold S., additional
- Published
- 2017
- Full Text
- View/download PDF
46. Identification of a Substrate-selective Exosite within the Metalloproteinase Anthrax Lethal Factor
- Author
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Goldberg, Allison B., primary, Cho, Eunice, additional, Miller, Chad J., additional, Lou, Hua Jane, additional, and Turk, Benjamin E., additional
- Published
- 2017
- Full Text
- View/download PDF
47. The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome
- Author
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Carmit Strauss, Zvi Hayouka, Gideon Coster, Assaf Friedler, Liron Argaman, Michael Brandeis, and Michal Goldberg
- Subjects
DNA repair ,DNA damage ,Molecular Sequence Data ,Cell Cycle Proteins ,Biochemistry ,Anaphase-Promoting Complex-Cyclosome ,Cell Line ,Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome ,CDC27 ,Radiation, Ionizing ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,CHEK1 ,Phosphorylation ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,biology ,Cell Cycle ,Nuclear Proteins ,Ubiquitin-Protein Ligase Complexes ,Cell Biology ,Cell cycle ,G2-M DNA damage checkpoint ,Molecular biology ,Protein Structure, Tertiary ,Ubiquitin ligase ,Cell biology ,MDC1 ,DNA-Binding Proteins ,Trans-Activators ,biology.protein - Abstract
MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.
- Published
- 2007
48. ATP-induced Structural Transitions in PAN, the Proteasome-regulatory ATPase Complex in Archaea
- Author
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Alfred L. Goldberg, Michael Groll, Andrew A. Horwitz, Ami Navon, Christian Reis, and David M. Smith
- Subjects
Proteasome Endopeptidase Complex ,Archaeal Proteins ,Methanococcus ,ATPase ,medicine.medical_treatment ,Amino Acid Motifs ,Molecular Conformation ,Biochemistry ,Adenosine Triphosphate ,ATP hydrolysis ,medicine ,Animals ,Humans ,Trypsin ,Nucleotide ,Molecular Biology ,Adenosine Triphosphatases ,chemistry.chemical_classification ,Protease ,Dose-Response Relationship, Drug ,biology ,Hydrolysis ,Walker motifs ,Cell Biology ,Proteasome complex ,Archaea ,AAA proteins ,Kinetics ,Proteasome ,chemistry ,biology.protein ,Endopeptidase K - Abstract
ATP binding to the PAN-ATPase complex in Archaea or the homologous 19 S protease-regulatory complex in eukaryotes induces association with the 20 S proteasome and opening of its substrate entry channel, whereas ATP hydrolysis allows unfolding of globular substrates. To clarify the conformational changes associated with ATP binding and hydrolysis, we used protease sensitivity to monitor the conformations of the PAN ATPase from Methanococcus jannischii. Exhaustive trypsin treatment of PAN generated five distinct fragments, two of which differed when a nucleotide (either ATP, ATP gamma S, or ADP) was bound. Surprisingly, the nucleotide concentrations altering protease sensitivity were much lower (K(a) 20-40 microm) than are required for ATP-dependent protein breakdown by the PAN-20S proteasome complex (K(m) approximately 300-500 microm). Unlike trypsin, proteinase K yielded several fragments that differed in the ATP gamma S and ADP-bound forms, and thus revealed conformational transitions associated with ATP hydrolysis. Mapping the fragments generated by each revealed that nucleotide binding and hydrolysis induce local conformational changes, affecting the Walker A and B nucleotide-binding motif, as well as global changes extending to its carboxyl terminus. The location and overlap of the fragments also suggest that the conformation of the six subunits is not identical, probably because they do not all bind ATP simultaneously. Partial nucleotide occupancy was supported by direct assays, which demonstrated that, at saturating conditions, only four nucleotides are bound to hexameric PAN. Using the protease protection maps, we modeled the conformational changes associated with ATP binding and hydrolysis in PAN based on the x-ray structures of the homologous AAA ATPase, HslU.
- Published
- 2007
49. Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43
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Alonso P. Moreno, Yongquan Shen, Gary S. Goldberg, Xun Li, P. Raaj Khusial, and Hitoshi Ichikawa
- Subjects
Cell signaling ,Cell Communication ,Biology ,Biochemistry ,Cell junction ,Focal adhesion ,Mice ,Animals ,Phosphorylation ,RNA, Small Interfering ,Molecular Biology ,Mice, Knockout ,Focal Adhesions ,Tumor Suppressor Proteins ,Gap junction ,Gap Junctions ,Cell Biology ,Cell biology ,Cell Transformation, Neoplastic ,Crk-Associated Substrate Protein ,src-Family Kinases ,Connexin 43 ,Tyrosine ,Tyrosine kinase ,Proto-oncogene tyrosine-protein kinase Src - Abstract
The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.
- Published
- 2007
50. Fc Engineering Approaches to Enhance the Agonism and Effector Functions of an Anti-OX40 Antibody
- Author
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Zhang, Di, primary, Goldberg, Monica V., additional, and Chiu, Mark L., additional
- Published
- 2016
- Full Text
- View/download PDF
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