Lactacystin, a Specific Inhibitor of the Proteasome, Inhibits Human Platelet Lysosomal Cathepsin A-like Enzyme

Ostrowska, H; Wojcik, Cezary; Ōmura, Satoshi; Worowski, K

doi:10.1006/bbrc.1997.6434

Cited by 102 publications

(56 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…25 However, 3-methyladenine was found to be clearly more potent in interfering with GFP-NBR1 degradation, showing that NBR1 is mainly an autophagic substrate in this reporter system. Different from GFP-p62 and GFP-NBR1, GFP-LC3B responded similarly to autophagy-and proteasome inhibitors.…”

Section: Resultsmentioning

confidence: 85%

A reporter cell system to monitor autophagy based on p62/SQSTM1

Larsen¹,

Lamark²,

Øvervatn³

et al. 2010

Autophagy

137

105

View full text Add to dashboard Cite

M acroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy, cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut-off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFPp62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform the best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knockdown of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that A reporter cell system to monitor autophagy based on p62/SQSTM1

show abstract

Section: Resultsmentioning

confidence: 85%

A reporter cell system to monitor autophagy based on p62/SQSTM1

Larsen¹,

Lamark²,

Øvervatn³

et al. 2010

Autophagy

137

105

View full text Add to dashboard Cite

show abstract

“…We found that the half-life of FAT10 is about 2 h and that the rapid degradation of FAT10 is sensitive to lactacystin. Although lactacystin is not an entirely monospecific inhibitor of the proteasome (35,36), it is fair to conclude that FAT10 is most likely degraded by the proteasome. This conclusion we have reached for the HA-tagged version of mouse FAT10 was also proposed by Liu et al (10), who found that IFN-␥ treatment of the B-cell lymphoma line JY with the fairly unspecific proteasome inhibitor Ac-LLN led to an accumulation of endogenously expressed human FAT10.…”

Section: Discussionmentioning

confidence: 99%

The Ubiquitin-like Protein FAT10 Forms Covalent Conjugates and Induces Apoptosis

Raasi

Schmidtke

Groettrup

2001

Journal of Biological Chemistry

151

182

View full text Add to dashboard Cite

FAT10 is a ubiquitin-like protein that is encoded in the major histocompatibility complex class I locus and is synergistically inducible with interferon-␥ and tumor necrosis factor ␣. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved diglycine motif at its carboxyl terminus commonly used in ubiquitin-like proteins for isopeptide linkage to conjugated proteins. We investigated the function of FAT10 by expressing murine FAT10 in a hemagglutinin-tagged wild type form as well as a diglycine-deficient mutant form in mouse fibroblasts in a tetracycline-repressible manner. FAT10 expression did not affect major histocompatibility complex class I cell surface expression or antigen presentation. However, we found that wild type but not mutant FAT10 caused apoptosis within 24 h of induction in a caspase-dependent manner as indicated by annexin V cell surface staining and DNA fragmentation. Wild type FAT10, but not its diglycine mutant, was covalently conjugated to thus far unidentified proteins, indicating that specific FAT10 activating and conjugating enzymes must be operative in unstimulated fibroblasts. Because FAT10 expression causes apoptosis and is inducible with tumor necrosis factor ␣, it may be functionally involved in the programmed cell death mediated by this cytokine.The covalent posttranslational modification of proteins is a versatile principle of determining the half-life, intracellular localization, and activity of proteins. In addition to modification by small molecules like orthophosphate, acetic acid, lipids, or sugars, the attachment of protein tags via isopeptide linkage to lysine residues in target proteins has recently been recognized as a frequently used and very diverse theme in cell biology. The prototype of such a protein tag is ubiquitin, which, when assembled as a polyubiquitin chain onto substrate proteins, can target these proteins to the 26S proteasome for degradation (1). Recently, it has become clear that the manner in which ubiquitin is linked in polyubiquitin chains defines the fate of the modified protein. The specific targeting signal for the proteasome pathway is a polyubiquitin chain that uses the K48 residue of the proximal ubiquitin as a target for further rounds of ubiquitination. The formation of K63-linked ubiquitin polymers, in contrast, does not lead to degradation but seems to play a role in DNA repair (2) and endocytosis (3). The conjugation of receptors with a single ubiquitin moiety can also serve as a signal for endocytosis (4), whereas in the case of histone H2B, monoubiquitination appears to regulate nucleosome function and cell division (5).The specificity of substrate selection and the mode of ubiquitin conjugation are determined by an enzymatic cascade required for the activation and specific transfer of ubiquitin. It consists of ubiquitin-activating enzyme (E1) 1 , which uses the energy of ATP to form a thioester linkage between a cysteine residue of the E1 enzyme and the carboxyl terminus of ubiquitin consisting of two ...

show abstract

“…Lactacystin has been used for several years to study the in vivo effects of proteasome inhibition because it was believed to be exclusively specific for the proteasome (43). The conclusiveness of these experiments must now be questioned, since recent evidence showed that a cathepsin A-like enzyme from lysosomes (44) and tripeptidyl peptidase II (18) were also inhibited by lactacystin. However, at a concentration of 1 M, which was used for modulation of Ag presentation in this study, tripeptidyl peptidase II is not inhibited, and even at 10 M lactacystin the inhibition is only 50%.…”

Section: Discussionmentioning

confidence: 99%

The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses

Schwarz

Giuli

Schmidtke

et al. 2000

The Journal of Immunology

View full text Add to dashboard Cite

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5–1 μM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89–168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5–1 μM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

show abstract

Lactacystin, a Specific Inhibitor of the Proteasome, Inhibits Human Platelet Lysosomal Cathepsin A-like Enzyme

Cited by 102 publications

References 20 publications

A reporter cell system to monitor autophagy based on p62/SQSTM1

A reporter cell system to monitor autophagy based on p62/SQSTM1

The Ubiquitin-like Protein FAT10 Forms Covalent Conjugates and Induces Apoptosis

The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses

Contact Info

Product

Resources

About