butabindide and alanyl-alanyl-phenylalanine-chloromethyl-ketone

Recent studies have identified Ca(2+) stores in sperm cells; however, it is not clear whether these Ca(2+) stores are functional and how they are mobilized. Here, in vitro and in vivo, we determined that tripeptidyl peptidase II antagonists strongly activated the cAMP/PKA signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation. We demonstrated that in the absence of Ca(2+), TPIII antagonists elevated the intracellular Ca(2+) levels in sperm, resulting in a marked improvement in sperm movement, capacitation, acrosome reaction, and the in vitro fertilizing ability. This antagonist-induced release of intracellular Ca(2+) could be blocked by the inhibitors of ryanodine receptors (RyRs) which are the main intracellular Ca(2+) channels responsible for releasing stored Ca(2+). Consistent with these results, indirect immunofluorescence assay using anti-RyR antibodies further validated the presence of RyR3 in the acrosomal region of mature sperm. Thus, TPPII can regulate sperm maturation by modulating intracellular Ca(2+) stores via the type 3 RyR.

Topics: Acrosome Reaction; Amino Acid Chloromethyl Ketones; Aminopeptidases; Animals; Blotting, Western; Calcium; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dantrolene; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Indoles; Intracellular Space; Male; Mice; Mice, Inbred C57BL; Muscle Relaxants, Central; Phosphorylation; Ryanodine; Ryanodine Receptor Calcium Release Channel; Serine Endopeptidases; Signal Transduction; Sperm Capacitation; Spermatozoa

A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of tripeptidyl peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.

Topics: Acids; Amino Acid Chloromethyl Ketones; Aminopeptidases; Antigen Presentation; Brefeldin A; Cell Line; Cell Line, Tumor; Coumarins; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Drug Stability; Histocompatibility Antigens Class I; HLA-B27 Antigen; Humans; Hydrogen-Ion Concentration; Indoles; Lymphocytes; Oligopeptides; Proteasome Endopeptidase Complex; RNA Interference; RNA, Small Interfering; Serine Endopeptidases; Serine Proteinase Inhibitors

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small proteins. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis. The pathological consequences of loss of activity are only manifested in neuronal cells suggesting that TPP-I may be involved in the lysosomal degradation of neuropeptides. We have investigated the degradation of the C-terminal octapeptide of sulphated cholecystokinin (CCK-8S) by a lysosomal fraction purified from mouse brain. Degradation products were characterised by reversed phase HPLC and mass spectrometry. Incubation of CCK-8S with brain lysosomes results in the sequential removal of the tripeptides DY(SO(3)H)M and Glycl-Tryptophanyl-Methionine from the N-terminus of CCK-8S. Degradation of CCK-8S in the isolated lysosomal fraction is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an inhibitor of TPP-I. Butabindide, a specific inhibitor of TPP-II, a cell surface peptidase which also cleaves CCK-8S, inhibits TPP-I but kinetic studies indicate that the Ki for inhibition of TPP-I is 1000-fold higher than the Ki for the inhibition of TPP-II. Consequently, higher concentrations of butabindide are required for the inhibition of CCK-8S degradation by TPP-I than by TPP-II. These results indicate that whereas cell surface TPP-II is responsible for regulating extracellular CCK-8S levels, lysosomal TPP-I is largely responsible for the degradation of CCK-8S which enters the cell by receptor-mediated endocytosis.

Topics: Amino Acid Chloromethyl Ketones; Aminopeptidases; Animals; Brain; Chromatography, High Pressure Liquid; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Enzyme Inhibitors; Indoles; Lysosomes; Mice; Serine Proteases; Sincalide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Tripeptidyl-Peptidase 1