Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors

Abstract

Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, β-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-014-0906-5) contains supplementary material, which is available to authorized users.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

“…Recently, a cDNA library was obtained from several potato cultivars and some proteins were heterologously expressed and tested against different classes of proteases. This work showed that inhibition of aspartic proteases is also possible in potato protease inhibitors belonging to group B (Fischer et al, 2015).…”

“…The 2F o -F c electron density maps of the E3Ad and E3Ad_N19D inhibitor structures clearly showed the presence of three disulfide bridges between positions Cys48-Cys93, Cys142-Cys158 and Cys148-Cys152. These disulfide bridges are present in all Kunitz-type STI family inhibitors able to inhibit aspartic proteases characterized to date, except those classified as part of the group B (Cater et al, 2002;Fischer et al, 2015;Keilová and Tomášek, 1976a;Mares et al, 1989).…”

“…Inhibitors of the Kunitz-type soybean trypsin inhibitor (STI) family are among the most versatile protease inhibitors reported, being able to interact with proteases belonging to different mechanistic classes like serine, aspartic and cysteine proteases, as well as other enzymes: a-amylase and sucrose invertase (Azarkan et al, 2011;Cater et al, 2002;Fischer et al, 2015;Franco et al, 2002;Glaczinski et al, 2002;Keilová and Tomášek, 1976a). These inhibitors have a b-trefoil fold, composed of 12 b-strands arranged in threefold pseudo-symmetry units or subdomains with the presence of helices in some cases (Broom et al, 2012;McLachlan, 1979;Murzin et al, 1992;Renko et al, 2012).…”

“…Although first studies on bifunctional inhibitors of the Kunitz-type STI family against aspartic proteases were performed using human cathepsin D (EC 3.4.23.5) (Keilová and Tomášek, 1976b), to date there is evidence that Kunitz-type STI family members are also able to inhibit other aspartic proteases of the clan AA, family A1, such as saccharopepsin (EC 3.4.21.41) (Cater et al, 2002), memapsin 1 (EC 3.4.23.45) (Fischer et al, 2015), and Plasmepsin II (EC 3.4.23.39) (this work).…”

Purificación, clonación y expresión de inhibidores peptídicos de proteasas de potencial aplicación biomédica a partir de <i>Solanum tuberosum</i> spp.

“…2011. El continuo avance en el conocimiento de su acción ha dado lugar a numerosos recientes reportes que expresan su potencial reemplazo de terapias con drogas sintéticas en la farmacología (Atanasov A G. 2015, Fischer 2015. Esperamos que la discusión de este trabajo resulte provechosa para el avance del estudio de esta clase de moléculas.…”