inhibitors from mushrooms
Peptidases are enzymes that cleave peptide
bonds; and are present in all organisms. They play
important roles in many physiological processes, from
digestion of food proteins to highly regulated cascades such as the
clotting cascade, programmed cell death pathways, immune response,
of hormones, and others. Given their important roles in cell and
physiology they are also involved in many pathological processes in
such as cancer, diabetes, muscular dystrophy, multiple sclerosis,
arthritis, osteoarthritis and Alzheimer’s disease. They are important
factors in many pathogenic bacteria, viruses, fungi and parasites.
regulation of proteolytic activity is vital and one of the most important
mechanisms of peptidase activity regulation
is interaction with specific inhibitors.
Peptidases and peptidase inhibitors are important targets and tools in
search for new compounds with applications in medicine, pharmaceutical
Our group is investigating inhibitors
of cysteine peptidases, inhibitors
of serine peptidases
and the corresponding peptidases
from our model mushrooms, clouded agaric (Clitocybe
nebularis), parasol mushroom (Macrolepiota
procera), inky cap mushroom (Coprinopsis
cinerea), and button mushroom (Agaricus
bisporus). Our main objective is the application of such proteins in
biotechnological processes and agriculture.
mushrooms growing in
Mycocypins – fungal cysteine peptidase
We have isolated a
cysteine peptidase inhibitor named clitocypin
from the mushroom clouded agaric (Clitocybe
nebularis). Because of its unique characteristics, a new
family, I48, has
been established for clitocypin in the MEROPS
classification, of which it is currently the only characterized member.
for similar cysteine peptidase inhibitors from mushrooms has led to
identification of a family of peptidase inhibitors named macrocypins
from the parasol mushroom (Macrolepiota procera).
A new family, I85, has been established in
classification for macrocypins. The characterization of clitocypin
and macrocypins at the genetic level has revealed that they are encoded
families of genes with greater sequence variability
that in macrocypins
affects their inhibitory profile. The inhibitory profiles of mycocypins
and macrocypins) include many papain-like cysteine peptidases (family
CA), legumain (family C13, clan CD) and the serine peptidase trypsin
clan PA). Determination of the crystal structure of mycocypins has
to identify the reactive sites responsible for inhibition of individual
of peptidases; the latter have been confirmed by mutagenesis.
Mycocypins, clitocypin (family I48) and macrocypins (family I85), share
a beta-trefoil fold.
- Sabotič J, Kilaru S, Budič M, Buh
Gašparič M, Gruden K, Bailey A, Foster GD, Kos J. Protease inhibitors
clitocypin and macrocypin are differentially expressed within
basidiomycete fruiting bodies. Biochimie (2011) 93 (10): 1685-1693.
- Renko M, Sabotič J, Mihelič M,
Brzin J, Kos J &
Turk D. Versatile loops in mycocypins
inhibit three protease families. J Biol Chem (2010) 285 (1):
J, Popovič T, Puizdar V & Brzin J. Macrocypins,
a family of cysteine protease
inhibitors from the basidiomycete Macrolepiota
procera. FEBS J (2009) 276 (16): 4334-4345
- Sabotič J, Galeša K, Popovič T,
Leonardi A & Brzin
J. Comparison of natural and recombinant
clitocypins, the fungal cysteine protease inhibitors. Protein
(2007) 53 (1): 104-111.
J, Gaser D, Rogelj B, Gruden K, Štrukelj B
& Brzin J. Heterogeneity in the
cysteine protease inhibitor clitocypin gene family. Biol Chem
K, Thomas RM, Kidrič M & Pain RH. Clitocypin,
a new cysteine proteinase
inhibitor, is monomeric: impact on the mechanism of folding.
Biophys Res Commun (2004) 324 (2): 576-578.
- Brzin J, Rogelj B, Popovič T,
Štrukelj B & Ritonja
A. Clitocypin, a new type of cysteine
proteinase inhibitor from fruit bodies of mushroom Clitocybe
nebularis. J Biol Chem (2000) 275 (26):
Fungal serine peptidase inhibitors
We have isolated serine peptidase
inhibitors CnSPI (Clitocybe nebularis
serine protease inhibitors) from the clouded agaric (C.
nebularis). One of them, named cnispin,
has been characterized in detail. It exhibits very
specific inhibition of trypsin. Functional analysis suggests a dual
role for cnispin, namely a regulatory role in the endogenous
and a defensive role against predatory insects. Cnispin is the first
inhibitor of the family I66 in the MEROPS
classification to be characterized at
the genetic, biochemical and functional levels. Characterization of a
similar peptidase inhibitor from the inky cap (Coprinopsis cinerea),
cospin, widened the knowledge on fungal trypsin inhibitors.
Determination of the three-dimensional structure enabled elucidation of
the molecular inhibitory mechanism of these trypsin-specific inhibitors
and revealed the incredible functional diversity of the beta-trefoil
- Sabotič J, Bleuler-Martinez S, Renko M,
Avanzo Caglič P, Kallert S, Štrukelj B, Turk D, Aebi M, Kos J, Künzler
M. Structural basis of trypsin inhibition and entomotoxicity of cospin,
a serine protease inhibitor involved in defence of Coprinopsis cinerea
fruiting bodies. J Biol Chem (2012) 287 (6): 3898-3907.
- Avanzo P, Sabotič J, Anžlovar S,
Popovič T, Leonardi
A, Pain RH, Kos J & Brzin J. Trypsin-specific
inhibitors from the basidiomycete Clitocybe
nebularis with regulatory and defensive functions.
Microbiology (2009) 155
Peptidases from mushrooms
investigated the hitherto poorly known proteolytic potential of
We have shown that all four major catalytic classes are present, with
prevailing. The occurrence of cysteine peptidase activity has been
for the first time in mushrooms. We have found unexpected diversity of
the majority of which are insensitive to standard class-specific
inhibitors. Some of them are inhibited only by endogenous fungal
inhibitors, namely cysteine peptidase inhibitor clitocypin and serine
inhibitors CnSPI from basidiomycete Clitocybe
have found unexpected diversity and a number of peptidases
that show characteristics indicating the uniqueness of proteolytic
fungal origin. Partial characterization of putative aspartic peptidases
clouded agaric, that showed sequence similarity to the A01.018 group of the
classification, have confirmed the diversity and riches of proteolytic
- Sabotič J, Trček T, Popovič T
& Brzin J. Basidiomycetes harbour a
hidden treasure of
proteolytic diversity. J Biotechnol (2007) 128 (2): 297-307
- Sabotič J, Popovič T &
Brzin J. Aspartic proteases from
basidiomycete Clitocybe nebularis.
(2009) 82 (4): 739-745
Applications of fungal peptidases and peptidase
research is oriented towards application of
peptidase inhibitors in crop protection. In collaboration with other
laboratories we are investigating their influence on growth and
different agricultural pests, including insects (Colorado potato
fly), slugs, moulds and bacteria.
- Istinič I, Buh Gašparič M, Sabotič J, Gruden K, Brzin J, Žel J. Uporaba makrocipinov kot pesticidnih učinkovin: številka prijave: P-201100304 z dne 10.8.2011. Ljubljana: Urad RS za intelektualno lastnino, 2011. 26 str.
We are also concerned with the application of
fungal peptidase inhibitors in cancer and the immune response.
enzymes play an important role in tumour progression and metastasis.
Mushroom-derived protein inhibitors of cysteine or serine peptidases
features of selectivity and specificity. They show mushrooms to be a
source of peptidase inhibitors that could serve as tools in research
an understanding of the role of individual peptidases at different
tumour progression, as well as in drug development and design for
immunomodulatory therapies of cancer patients.
Open access book Affinity Chromatography (Intech, 2012) with our chapter "The Value of Fungal Protease Inhibitors in Affinity Chromatography", that describes the
application of fungal peptidase inhibitors as ligands in affinity
chromatography using different supports for isolation of different