Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase.

Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.

Subcellular localization of nardilysin during mouse oocyte maturation.

We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with beta-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This finding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.

Nuclear shuttling of the peptidase nardilysin.

The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain.

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.

Nardilysin cleaves peptides at monobasic sites.

Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.

The use of proteolysis to study the structure of nardilysin.

Treatment of a 128 kDa mouse nardilysin with trypsin initially produced an active 105 kDa N-terminally cleaved form. Continued trypsin digestion occurred at the C-terminus, producing inactive core species of approximately 92, 76.5, and 62 kDa. Protease V8 digestion generated a stable approximately 105 kDa form, nardilysin(V8), that was cleaved near the N-terminal trypsin site. The approximately 105 kDa nardilysin(V8) exhibited the same K(m) as did the uncleaved enzyme for substrates of the type Abz-GGFX(1)X(2)X(3)VGQ-EDDnp, where X residues were varied. However, k(cat) for nardilysin(V8) was 5-6 times greater. Both uncleaved nardilysin and nardilysin(V8) are inhibited by NaCl; however, nardilysin(V8) exhibits an IC(50) of approximately 2 mM compared to an IC(50) of approximately 50 mM for uncleaved nardilysin. Nardilysin(V8) is more sensitive to inhibition by phosphate buffer. Treatment of nardilysin(V8) with trypsin generated primarily the 92 kDa form which was inactive. Attempts to express nardilysin as a 105 kDa truncated N-terminal form or as a C-terminally truncated form led to inactive proteins.