benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and pepstatin

The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation.. The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.. Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.. Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.

Topics: Animals; cis-trans-Isomerases; Cone Opsins; Cysteine Proteinase Inhibitors; Diterpenes; Eye; Leucine; Leupeptins; Lysosomes; Mice; Mice, Knockout; Organ Culture Techniques; Pepstatins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Real-Time Polymerase Chain Reaction; Retinaldehyde; Transcription, Genetic

TAR DNA-binding protein-43 (TDP-43) is a nuclear protein functioning in the regulation of transcription and mRNA splicing. TDP-43 is accumulated in ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) diseased brains. However, the pathways involved in the clearance of TDP-43 and its pathogenic form (TDP-25), a truncated form of TDP-43, are still not elucidated. In this study, we demonstrated that the protein levels of TDP-43 and TDP-25 were increased in cells treated with a proteasome inhibitor, MG132, or an autophagy inhibitor, 3-MA, whereas, they were decreased in cells treated with an enhancer of autophagy, trehalose. Furthermore, more protein level changes of TDP-25 than TDP-43 were observed in cells treated with above inhibitors or enhancer. Thus, our data suggest that TDP-43 and TDP-25 are degraded by both proteasome and autophagy with TDP-25 being more regulated.

Topics: Adenine; Autophagy; Cell Line; DNA-Binding Proteins; Humans; Leupeptins; Pepstatins; Peptide Fragments; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Trehalose; Ubiquitin

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent gamma-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by gamma-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PS1 is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/gamma-secretase activity in the functional regulation of adhesion molecules.

Topics: Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Carbamates; Cell Membrane; Central Nervous System Neoplasms; Dipeptides; Embryo, Mammalian; Endopeptidases; Fibroblasts; Glioma; Humans; Hyaluronan Receptors; Leupeptins; Membrane Proteins; Mice; Mice, Knockout; Pepstatins; Presenilin-1; Protease Inhibitors; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Topics: Aged; Alkaloids; Amino Acid Chloromethyl Ketones; Aprotinin; Benzophenanthridines; Benzyl Compounds; Caspase 3; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; DNA; DNA Fragmentation; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Leucine; Leupeptins; Male; Pepstatins; Phenanthridines; Protein Kinase C; Pyridines; Tumor Cells, Cultured

Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS1 and PS2). Several lines of evidence indicate that PS1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome.. We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses.. We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated that two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of APP alpha and A beta elicited by CTF-PS1/PS2.. Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP maturation upstream to alpha-and beta/gamma-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular concentration of CTFs.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Glycopeptides; Humans; Leucine; Leupeptins; Membrane Proteins; Multienzyme Complexes; Oligopeptides; Pepstatins; Presenilin-1; Presenilin-2; Proteasome Endopeptidase Complex; Recombinant Proteins; Sulfones; Transfection