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Peptidase 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 simple digestion of food proteins to highly regulated cascades such as the blood clotting cascade, programmed cell death pathways, immune response, activation of hormones, and others. Given their important roles in cell and organism physiology they are also involved in many pathological processes in humans, such as cancer, diabetes, muscular dystrophy, multiple sclerosis, rheumatoid arthritis, osteoarthritis and Alzheimer’s disease. They are important virulence factors in many pathogenic bacteria, viruses, fungi and parasites. Therefore, 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 the search for new compounds with applications in medicine, pharmaceutical industry and agriculture.

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 medicine, biotechnological processes and agriculture.

C nebularis
M procera C cinerea
Clouded agaric mushroom Parasol mushroom Inkycap mushrooms growing in
Petri dish

Mycocypins – fungal cysteine peptidase inhibitors

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 peptidase inhibitors classification, of which it is currently the only characterized member. The search 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 the MEROPS classification for macrocypins. The characterization of clitocypin and macrocypins at the genetic level has revealed that they are encoded by families of genes with greater sequence variability that in macrocypins affects their inhibitory profile. The inhibitory profiles of mycocypins (clitocypin and macrocypins) include many papain-like cysteine peptidases (family C1, clan CA), legumain (family C13, clan CD) and the serine peptidase trypsin (family S1, clan PA). Determination of the crystal structure of mycocypins has enabled us to identify the reactive sites responsible for inhibition of individual types of peptidases; the latter have been confirmed by mutagenesis. Mycocypins, clitocypin (family I48) and macrocypins (family I85), share a beta-trefoil fold.

3D klitocipin 3D kompleks 3D makrocipin

Selected publications:


  • 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): 308-316.
  • Sabotič 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 Expr Purif (2007) 53 (1): 104-111. 
  • Sabotič J, Gaser D, Rogelj B, Gruden K, Štrukelj B & Brzin J. Heterogeneity in the cysteine protease inhibitor clitocypin gene family. Biol Chem (2006) 387 (12): 1559-1566.
  • Galeša K, Thomas RM, Kidrič M & Pain RH. Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of folding. Biochem 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): 20104-20109.

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 biological role for cnispin, namely a regulatory role in the endogenous proteolytic system and a defensive role against predatory insects. Cnispin is the first serine peptidase 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 fold loops.

Selected publications:

  • 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 (Pt 12):3971-3981.

Peptidases from mushrooms

We have investigated the hitherto poorly known proteolytic potential of basidiomycetes. We have shown that all four major catalytic classes are present, with serine peptidases prevailing. The occurrence of cysteine peptidase activity has been demonstrated for the first time in mushrooms. We have found unexpected diversity of peptidases, the majority of which are insensitive to standard class-specific peptidase inhibitors. Some of them are inhibited only by endogenous fungal peptidase inhibitors, namely cysteine peptidase inhibitor clitocypin and serine peptidase inhibitors CnSPI from basidiomycete Clitocybe nebularis. We have found unexpected diversity and a number of peptidases that show characteristics indicating the uniqueness of proteolytic enzymes of fungal origin. Partial characterization of putative aspartic peptidases from clouded agaric, that showed sequence similarity to the A01.018 group of the MEROPS classification, have confirmed the diversity and riches of proteolytic enzymes in mushrooms.

Selected publications:

  • 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. Croat Chem Acta (2009) 82 (4): 739-745

Applications of fungal peptidases and peptidase inhibitors

Our research is oriented towards application of peptidase inhibitors in crop protection. In collaboration with other laboratories we are investigating their influence on growth and development of different agricultural pests, including insects (Colorado potato beetle, fruit fly), slugs, moulds and bacteria.

Patent application:

  • 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. Proteolytic enzymes play an important role in tumour progression and metastasis. Mushroom-derived protein inhibitors of cysteine or serine peptidases exhibit unique features of selectivity and specificity. They show mushrooms to be a valuable source of peptidase inhibitors that could serve as tools in research leading to an understanding of the role of individual peptidases at different stages of tumour progression, as well as in drug development and design for anti-tumour and immunomodulatory therapies of cancer patients.

kol hrosc hife PC12

Affinity chromatography

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 peptidases.

Contact:

Jerica Sabotič

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