The cysteine protease inhibition by cystatins. The results

The
majority of the proteins present in sesame seeds are storage proteins
categorized as albumins (9%), globulins (67%), prolamins (1%) and glutelins
(7%) on the basis of their solubility. The water-insoluble 11S globulin and the
soluble 2S albumin are the two major storage proteins of sesame (Rivas R et al., 1981). Seeds development and germination processes
is mainly affected by proteolysis process. Proteolysis is a metabolic pathway
that involves comprehensive metabolic networks, different subcellular
partitions and types of proteases, mostly cysteine-, serine-, aspartic- and
metallo-proteases (van der Hoorn, 2008). Among the approximately 800 proteases
encoded by plant genomes, more than 140 correspond to cysteine proteases (CP) (Rawlings et al., 2010). The
main physiological role of CPs is metabolic degradation of peptides and
proteins (Storer and Menard, 1994).

Papain-like
cysteine protease reported to play an
important role in a process of seed germination by hydrolysing seed storage
proteins (Tsuji et al., 2013). Several
cysteine proteinase genes also involved in plant defense response against fungus
(Höwing et al., 2014), viruz (Adenot et al., 2006), and bacteria (Bernoux et
al., 2008). Despite important role of cysteine endopeptidases, their
uncontrolled activity can induce pathology and the destruction of tissue
structures (Szewi?ska et al., 2016). In order to prevent such damage from excess
activity, the regulatory mechanism has been developed by many organisms at the
level of modified transcription, translation and post-translation synthesized
by cells, in the form of specific protease inhibitors (Arai et al., 2002). In this role, cystatins are known as
specific and natural inhibitors of papain-like cysteine proteases C1A family
act as regulators of harmful cysteine proteases activities (Viswanathan et al.
2011). Plant cystatins
(phytocystatins) are assumed to regulate endogenous cysteine protease
activities in response to biotic and abiotic stresses during seed development
and germination, when stored reserves are mobilized for embryogenesis and
seedling growth (Abe et
al., 1987; Callis, 1995). They
were also implicated in programmed cell death by modulating cysteine protease
activity in the regulation of protein turnover (Solomon
et al., 1999; Xu and Chye, 1999).

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Numerous spectroscopic, kinetic, and
crystallographic studies have been carried out to explain the mechanism of
cysteine protease inhibition by cystatins. The results have shown that the
inhibitor binds in a one-step process that is simple, reversible, and
second-order type. In addition, those studies have revealed that enzymes with a
blocked active centre could still bind cystatins, albeit with lower affinity (Bode et al., 1990). This indicates that cysteine
protease–cystatin interactions are not based on a simple reaction with the
catalytic cysteine residue of the enzyme, as is typical of substrates, but that
they consist of hydrophobic contacts between the binding regions of cystatins
and the corresponding residues forming the binding pockets of the enzyme.
Despite their structural homology and similar mode of inhibition, cystatins
display quite different enzyme affinities (Abrahamson, 1994).

In
this study, Sesamum indicum cysteine
protease (SiCP) have been molecularly characterized and comparative sequence alignment
have analysed related with functional motifs. The recombinant proteins could successfully purify as a soluble and
active protein in a prokaryotic expression system. The possible affinities interaction of cystatins in the control of SiCP
activity during sesame seed germination is discussed.