benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and 4-(2-aminoethyl)benzenesulfonylfluoride

Interactions between viruses and their host cells are important determinants of virus replication and of immune responses to the virus. However, these interactions and resulting consequences of these interactions remain poorly defined. Numerous recent quantitative proteomic approaches have measured host proteins affected by virus infection. Here, we used activity-based protein profiling (ABPP) to measure functional alterations in host serine hydrolases after influenza A virus infection of Madin-Darby canine kidney and human A549 lung cells. We identified 62 serine proteases. We then combined the ABPP approach with stable isotope labeling to directly measure how serine hydrolase activities were affected by virus infection. Differentially regulated SHs mapped into a few key cellular pathway systems, most notably the proteasomal system. The specific serine protease inhibitors Aprotinin and Pefablock and specific proteasomal inhibitors Bortezomib and MG132 significantly inhibited influenza virus growth. Some inhibitors also down-regulated activities of several proteasomal proteins, including PSMA1, PSMA2, and PMSB3. Genetic knockdown of PMSA2 also attenuated influenza virus replication. These findings further our understanding of enzymatic cellular processes affected by influenza virus and may be beneficial in the search for additional antiviral therapeutic targets.

Topics: Animals; Aprotinin; Blotting, Western; Boronic Acids; Bortezomib; Cell Line, Tumor; Dogs; Host-Pathogen Interactions; Humans; Influenza A Virus, H1N1 Subtype; Leupeptins; Madin Darby Canine Kidney Cells; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteome; Proteomics; Pyrazines; RNA Interference; Serine Proteases; Sulfones; Up-Regulation; Virus Replication

The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.

Topics: Androgen Receptor Antagonists; Androgens; Anilides; Antineoplastic Agents; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Male; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Serine Proteinase Inhibitors; Sulfones; Tosyl Compounds

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