Nostocyclopeptides (Ncps) are a small class of bioactive nonribosomal peptides thus far identified only in cyanobacteria of the genus Nostoc. They are composed of six-seven amino acid residues and contain a unique imino linkage formed between C-terminal aldehyde and an N-terminal amine group of the conserved tyrosine. Nostocyclopeptides occur both in cyclic and linear form.
1. Introduction
The 26S proteasome is a large (2.4 MDa), multifunctional and ATP-dependent enzymatic complex with chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities
[1][2][3][4]. In eukaryotic organisms, it recognizes and degrades proteins with covalently attached ubiquitin (8.5 kDa protein)
[5][6]. The 26S proteasome is composed of a 20S barrel-shaped core particle (700 kDa) responsible for proteolytic activity and one or two 19S (890 kDa) regulatory subunits with ubiquitin-binding sites
[3][4][7]. The 20S proteasome also occurs as a free complex that degrades proteins in the ubiquitin-independent pathway
[8][9]. In humans, the dysfunction of this proteolytic machinery leads to changes in protein profile and, ultimately, to serious health problems. Therefore, proteasome regulators are explored as promising therapeutic agents for a range of diseases (e.g., cancer, autoimmune disorders, inflammation, malaria)
[10][11][12]. The majority of the known 20S proteasome inhibitors belongs to peptide-based structures such as peptide aldehydes, boronates, epoxyketones, or peptide vinyl sulfones
[13][14][15]. Some of the active compounds are of natural origin. Leupeptin, isolated from several strains of Gram-positive bacteria of the order
Actinomycetales, inhibits T-L activity of the 20S proteasome
[16]. Tyropeptin A, a peptide aldehyde produced by the soil
Streptomycetales of the genus
Kitasatospora, strain MK993-dF2, inhibits mainly CT-L activity
[17][18]. Marine fungus
Peicillium fellutanum is a producer of fellutamide B, a strong inhibitor of CT-L activity (IC
50 9.4 nM) with mild effects on T-L (IC
50 2.0 μM) and C-L (IC
50 1.2 μM) activities
[19]. The proteasome inhibition within the nanomolar to the micromolar range of IC
50 was also documented for metabolites isolated from cyanobacteria
Symploca sp.,
Scytonema hofmannii, and
Nostoc.
In our preliminary studies, fractions from
Nostoc edaphicum Nostoc edaphicum CCNP1411 containing
nostocyclopeptides (Ncps) inhibited the chymotrypsin-like activity of the 20S proteasome.
Ncps constitute a small group of nonribosomal peptides solely produced by cyanobacteria
of the genus
Nostoc Nostoc. The biological activity of the peptides was reported in several studies.
According to Golakoti et al.
[20], Ncp-A1 and Ncp-A2 have cytotoxic activity against
human colorectal adenocarcinoma (LoVo) and human nasopharyngeal (KB) cell lines
(IC
50 ca. 1
μµM). Another nostocyclopeptide variant, Ncp-M1, was shown to inhibit the
transport of toxic microcystin-LR and nodularin into hepatocytes by blocking organic
anion transporter polypeptides, OATP1B1, and OATP1B3. These polypeptides are also
overexpressed in cancer cells
[21][22]. The role of Ncp-M1 and its analogs as antitumor
agents and as tools to study membrane transport was proposed
[21][23][24].
2. NoRestocyclopeptides as New Inhibitors of 20S Proteasomeults and Discussion
Thus far, the presence of Ncps was reported in five strains of
Nostoc isolated from different habitats
[20][21][22][23][24]. This includes two Baltic strains: XSPORK 13A producing the cyclic Ncp-M1
[21] and CCNP1411 producing 10 other Ncps variants
[24]. The putative structures of the five linear and five cyclic Ncps variants produced by CCNP1411 were elucidated based on mass spectra fragmentation patterns
[24]. Two of the cyclic forms, Ncp-A1 and Ncp-A2, enclosed by imino linkage between the
N-terminal amine group of conserved Tyr and
C-terminal aldehyde group of Leu or Phe, were previously identified in
Nostoc sp. ATCC53789 isolated from lichen
[20]. In position 6 of the Ncps from CCNP1411, 4-methylproline (MePro) or Pro is present, while Ile or Val is in position 4 (
Figure 1). In the current study, we were able to isolate 6 out of 10 Ncps produced by CCNP1411 (
Table 1): three cyclic variants (Ncp-A1, Ncp-A2, and Ncp-E2), two linear aldehyde forms of the cyclic variants (Ncp-A2-L and Ncp-E2-L), and the six-amino acid peptide Ncp-E4-L lacking the aldehyde group in the
C-terminus. In the case of four other Ncps produced by CCNP1411 (Ncp-A1-L, Ncp-E1, Ncp-E1-L, Ncp-E3), their purity and/or quantities were not sufficient for inclusion in the study. Ncp-A2-L (
Figure 1) was the only variant obtained in sufficient amounts for NMR analyses. The
1H NMR spectrum of Ncp-A2-L displayed a typical pattern of a peptide. The COSY, TOCSY, and HMBC experiments allowed for the identification of the residues in Ncp-A2-L as Tyr, Gly, Gln, Ile, Ser, MePro, and phenylalaninal (Phe-H) (
Table 2,
Figure 1,
Figures S1–S5). The amino acid sequence was confirmed by TOCSY data. The presence of two aromatic amino acid residues was recognized by the signals occurring in the aromatic region of the spectrum (δ
H 6.78–7.26 ppm). One of them was identified as tyrosine-based on the AA’BB’ spin system between the aromatic protons (Tyr-H5/5′ and Tyr-H6/6′, JH, H = 8.0 Hz). The second aromatic residue was identified as phenylalanine based on the TOCSY interaction between 34, 35, and 36 protons and the HMBC correlation from two diastereotopic methylene protons 32a (δ
H 2.57 ppm) and 32b (δ
H 2.98 ppm) to the aromatic 34/34′ carbons (
Figures S3 and S5). The 4-methyl group of the proline residue was identified based on the
1H NMR doublet signal at δ 0.82 ppm (protons 29) and the HMBC correlation between the methyl protons with 26 (δ
C 37.3 ppm) and 28 (δ
C 55.0 ppm) carbons (
Figures S1 and S5). The signal at δ
H 9.46 ppm was assigned to phenylalaninal aldehyde proton. The occurrence of the studied compound in the linear form was further confirmed by the lack of the ROESY correlation between tyrosine and phenylalanine residues.
Figure 1. ROESY and HMBC correlations in nostocyclopeptide Ncp-A2-L.
Table 1. Structures of six nostocyclopeptide-variants isolated from Nostoc edaphicum CCNP1411 as pure compounds.
Peptide Name |
Molecular Mass |
Structure |
Ncp-A1 |
+MePro |
6 |
Table 2. Nuclear Magnetic Resonance (NMR) Spectroscopic Data for Ncp-A2-L (Tyr-Gly-Gln-Ile-Ser-MePro-Phe-H).
Residue |
Position |
δC, type |
δH (J in Hz) |
ROESY |
HMBC a |
756 |
[Tyr | 1+Gly2+Gln3+Ile4+Ser5+MePro6+Leu7] |
Ncp-A2 |
Ncp-A2-L |
Tyr |
1 |
|
|
|
|
2 | 790 |
[Tyr1+Gly2+Gln3+Ile4+Ser5+MePro6+Phe7] |
169.9, C |
|
|
|
808 |
Tyr | 1+Gly2+Gln3+Ile4+Ser5+MePro6+Phe-H |
3 | 7 |
54.6, CH |
4.13, t (6.9, 6.9) |
NH(1), 6 |
|
Ncp-E2 |
742 |
2[Tyr1+Gly2+Gln3+Ile4+Ser5+Pro6+Leu7] |
4 |
36.0, CH |
3.04, dd (7.3, 12.9) |
6 |
|
Ncp-E2-L |
760 |
Tyr1+Gly2+Gln3+Ile4+Ser5+Pro6+Leu-H7 |
5/5′ |
125.5, C |
|
|
|
Ncp-E4-L |
676 |
Tyr1+Gly2+Gln3+Ile4+Ser5 |
6/6′ |
130.9, CH |
6.78, d (8.0) |
|
2, 4, 5 |
7 |
115.9, CH |
7.04, d (8.0) |
2, 3 |
|
NH2 |
155.3, C |
|
|
|
OH |
|
|
|
|
Gly |
8 |
170.7, C |
|
|
|
9 |
42.4, CH2 |
3.84, m |
NH(2) |
|
NH(1) |
|
8.46, t (5.6, 5.6) |
2 |
1 |
Gln |
10 |
|
|
|
|
11 |
173.1, C |
|
NH(3) |
|
12a |
53.2, CH |
4.30, m |
|
10 |
12b |
27.2, CH2 |
1.88, m |
|
|
13 |
|
1.99, m |
|
|
14 |
31.1, CH2 |
2.26, t (7.3, 7.3) |
|
11, 12, 14 |
NH(2) |
178.0, C |
|
9 |
|
NH2 |
|
8.25, d (7.6) |
|
8 |
Ile |
15 |
173.4, C |
|
|
|
16 |
58.2, CH |
4.09, t (8.1, 8.1) |
NH(4) |
17 |
17 |
36.0, CH |
1.77, m |
|
|
18 |
14.7, CH3 |
1.08, d (6.6) |
|
|
19 |
24.6, CH2 |
1.32, m |
|
|
20 |
10.0, CH3 |
0.79, t (7.3, 7.3) |
22 |
17, 19 |
NH(3) |
|
8.21, d (6.8) |
11 |
10 |
Ser |
21 |
|
|
|
|
22 |
n.o. |
|
|
|
23a |
n.o. |
4.59, m |
20 |
|
23b |
61.0, CH2 |
3.69, m |
|
|
NH(4) |
|
3.77, m |
|
|
OH |
|
8.31, d (5.1) |
16 |
15 |
MePro |
24 |
173.8, C |
|
|
|
25 |
61.3, CH |
4.15, dd (8.1, 9.3) |
|
24 |
26 |
37.3, CH2 |
2.13, m |
|
|
27 |
33.1, CH |
2.04, m |
|
|
28a |
55.0, CH2 |
2.84, t (10.5, 10.5) |
NH(5) |
|
28b |
|
3.86, m |
|
26 |
29 |
15.2, CH3 |
0.82, d (6.6) |
|
26, 28 |
Phe-H |
30 |
|
9.46, s |
|
|
31 |
n.o. |
4.01, m |
35 |
|
32a |
55.5, CH |
2.57, dd (10.6, 14.0) |
34 |
34/34′ |
32b |
34.2, CH2 |
2.98, dd (4.0, 14.5) |
|
|
33 |
|
|
32 |
|
34/34′ |
137.9, C |
7.26, m |
|
|
35/35′ |
129.5, CH |
7.15, d (7.2) |
31 |
|
36 |
128.7, CH |
7.18, m |
|
|
NH(5) |
126.6, CH |
7.46, d (9.3) |
25 |
24 |