ATP-Dependent Protease Complexes − Rakyat Biologi

ATP-dependent protease complexes are large, oligomeric complexes composed of chaperone and proteolytic subunits (Clp and proteasome families). In some cases the chaperone and proteolytic parts are located in the same polypeptide chain (Lon and FtsH families). These proteases are built on the principle of self-compartmentalization – the proteolytic active sites are buried inside a narrow channel in the oligomeric structure; only substrates unfolded and translocated inside by the chaperone part are accessible to degradation, thus the surrounding native proteins are protected from breakdown (Schmidt et al., 1999). This structural organization, in which the protease active sites are hidden into the interior chamber, allows only processive degradation of unfolded polypeptide chains - gradual degradation to small peptides (5-10 amino acids) and to amino acids, coupled to ATP-dependent, chaperone-assisted unfolding and translocation of the unfolded polypeptide.

Energy-dependent proteases, in cooperation with the chaperone system, act as protein quality control systems in maintaining the functional state of cell proteins in essential compartments such as cytosol, endoplasmic reticulum, mitochondria and chloroplasts (Leidhold and Voos 2007). As peptide bond hydrolysis is an exergonic reaction, ATP hydrolysis is most probably linked to specific recognition, regulation and control over proteolysis. Besides ATP coupling, these proteases are also able to degrade poorly structured or oxidatively modified proteins in an ATP-independent manner (Kurepa et al., 2009).

The Proteasome

The main proteolytic degradation system of eukaryotes, operating in the cytoplasm and nucleus, is the ubiquitin/26S proteasome pathway. The proteasome plays a crucial role in the turnover of regulatory proteins, cellular house-keeping and stress tolerance (Kurepa and Smalle, 2008). The structure and activities of proteasomes are highly conserved among eukaryotes, suggesting essential functions in protein homeostasis. This system is an extremely large and complex route for protein degradation, accounting for nearly 6% of the Arabidopsis thaliana transcriptome (Vierstra, 2009).

Proteins targeted for degradation are covalently tagged with the highly conserved 76 residue peptide ubiquitin (Ub) by a cascade of three types of enzymes ‒ E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) ‒ that catalyse the conjugation of Ub monomer to a lysine residue of the target protein. E3 controls the specificity of Ub conjugation. In the Arabidopsis genome, 2 E1s, at least 37 E2s and over 1,400 different E3s are expressed (Vierstra, 2009). Ub conjugation is a reversible reaction - detachment of Ub is catalysed by Ub-specific proteases that form a group of multiple enzymes, controlling ubiquitination and recycling Ub for reuse. While mono-ubiquitination of proteins facilitates interaction with ubiquitin binding domains in specific target proteins and has a regulatory function, polyubiquitinated proteins (at least four Ubs linked to target proteins by the residue lysine 48 of Ub) are recognized by specific receptors within the 26S proteasome or by adaptor proteins associated with the proteasome and are degraded in an ATP-dependent manner (Miura and Hasegava, 2010).

The 26S proteasome controls proteolysis of key components of numerous signalling pathways, regulated proteolysis of functional proteins and removal of misfolded and damaged proteins (Smalle and Vierstra, 2004; Vierstra, 2009). Besides ubiquitin, there is a set of other ubiquitin-like proteins involved in posttranslational modification of proteins such as RUB1/Nedd8, SUMO, HUB, ISG15, and ATG (Catala et al., 2007). SUMO (small ubiquitin-related modifiers) tagging of proteins proceeds in a pathway similar to that for ubiquitination, with sequential action of analogous E1, E2 and E3 enzymes. SUMO-specific proteases cleave off SUMO for re-use (Novatchkova et al., 2004). SUMOylation regulates protein degradation and localization, protein–protein interactions, transcriptional activity and counteracts the Ub-mediated degradation by the proteasome (Novatchkova et al., 2004).

The 26S proteasome is a 32-subunit, multicatalytic protease that degrades polyubiquitinated protein targets (Smalle and Vierstra 2004; Vierstra, 2009). It is composed of two subcomplexes - the barrel-shaped, proteolytically active core 20S proteasome (about 700 kDa in size) and the 19S regulatory particle (700 kDa) that recognizes, unfolds and channels Ub-targeted proteins into the 20S proteasome for degradation (Kurepa and Smalle, 2008). In the absence of ATP the 26S proteasome dissociates into 20S and 19S subcomplexes (Vierstra, 1996). The proteolytic core belongs to the threonine-class of proteases and possesses trypsin-like, chymotrypsin-like and peptidyl glutamyl-peptide-hydrolase activities. The core particle is built of four rings: two inner rings of seven beta-subunits each, three of them proteolytically active (the proteolytic chamber), and two outer rings of seven alpha-subunits (the entrance to the proteolytic chamber) which interact with the regulatory particle. The regulatory particle contains two subcomplexes, the lid and the base. The base contains six different AAA ATPases that form a ring-like structure and uses ATP hydrolysis to unfold target proteins, together with non-ATPase subunits that function as docking sites for proteins. The lid, an 8-subunit, non-ATPase assembly linked to the core particle by the base, is required for ubiquitin-conjugate degradation and is closely related to the signalosome (Schmidt et al., 1999). The structural composition of the proteasome is heterogeneous and some subunits are subjected to phosphorylation, acetylation and other post-translational modifications. The main functions of Ub-dependent proteolysis are degradation of misfolded or damaged proteins and of inherently unstable proteins that carry specific degradation signals, quality control by removal of proteins with translational errors, and inactivation of regulatory proteins. Phosphorylation and dephosphorylation in the signal transduction cascades often alter protein stability and affect the affinity of E3 for the respective target proteins. Further, the proteasome exerts control on metabolic fluxes by degrading key enzymes whose stability may depend on changes in metabolite concentrations (Kurepa and Smalle, 2008). Ub-independent proteolysis is involved in degradation of oxidized proteins - thus 20S may play an important role in tolerance to the secondary oxidative stress accompanying many primary abiotic stresses. In addition, plant proteasomes have RNAse activity, which is important in protection against pathogenic viruses.

Rotease Complexes in Chloroplasts and Mitochondria

Chloroplasts and mitochondria contain their own proteolytic systems, which are less complex than the ubiquitin/26S proteasome pathway. They are homologous to the bacterial proteases and have evolved in plants from single genes into multigene families (Adam, 2007; Janska et al., 2010). In Arabidopsis chloroplasts, at least 11 different types of protease families, encoded by more than 50 genes, have been found, amounting to about 2.3% of the chloroplast proteins (Sakamoto, 2006). The Clp complex is an abundant proteolytic system in chloroplast stroma. Its proteolytic subunits are encoded in Arabidopsis by 6 different genes, one of the products being targeted to mitochondria and 5 to chloroplasts. One of these genes is in the plastome and the others in the nuclear genome. Additional genes code for 4 non-catalytic ClpP-related ClpR subunits. The chaperone part of Clp in Arabidopsis is coded by three genes for chloroplasts (ClpC1, ClpC2, ClpD) and three for mitochondria (ClpX). There are an additional two copies of ClpS (unique for land plants) and one ClpT copy (Adam, 2007).

The structure of the Clp protease complex resembles that of the 26S proteasome. It consists of two functional elements: one, a barrel-shaped, hetero-oligomeric proteolytic core complex of 325-350 kDa, composed of two stacked heptameric rings of ClpP, ClpR and ClpS subunits with narrow (about 10 Ǻ) axial openings, forming a chamber which hides inside serine-type proteolytic active sites (catalytic triad Ser-His-Asp), and the other, two hexameric rings at the openings of the proteolytic core, composed of chaperones of the AAA+ superfamily of ATPases (Clp/Hsp100). The mitochondrial Clp core complex is a 320 kDa homotetradecamer of ClpP2 associated with ClpX chaperones (Peltier et al., 2004). ClpC and ClpX chaperones appear to be constitutively expressed, while ClpD is responsive to drought stress and senescence (Sakamoto, 2006).

Plant organelles also have FtsH and Lon ATP-dependent proteases in which the proteolytic active site and chaperone domain are parts of the same polypeptide chain. The FtsH gene family in Arabidopsis contains 12 members, 4 of whose products are targeted to mitochondria and 9 to chloroplasts. FtsH proteases have a 400-450 kDa hexameric ring-like structure formed by identical or closely related subunits. These provide an internal channel harbouring the proteolytic sites, access to which is controlled by the chaperone part. Each 74 kDa FtsH monomer consists of two trans-membrane helices at the N-terminus which anchor the protein to membranes. The stroma-facing AAA+ATPase domain and the proteolytic domain possess a conserved zinc-binding motif (His-Glu-X-X-His) at the C-terminus, typical of metalloproteases. FtsH complexes are heteromeric and its subunits are partially functionally redundant (Sakamoto, 2006).

Arabidopsis possesses four genes for Lon protease which are targeted to mitochondria, chloroplasts and peroxisomes (Adam, 2007; Janska et al., 2010). Lon is a hexameric ATP-dependent serine protease with a Ser-Lys dyad in the active site. Chaperone and catalytic domains are located in the same polypeptide.

The interesting hexameric DegP serine protease, with 16 paralogs, is coded for in the Arabidopsis genome. It is not involved in the ATP-dependent proteolytic pathway but switches between chaperoning and proteolytic activity under the influence of high temperature (Adam, 2007). The DegP monomers of 48 kDa are composed of a proteolytic domain at the N-terminus with the catalytic triad Ser-His-Asp, and two tandem PDZ domains at the C-terminus which regulate protein-protein interactions and recognition of the substrate proteins. In the chaperone form the active site of the protease is blocked by an auto-inhibitory segment. The temperature-induced conformational change pulls apart this segment, making the active centre accessible to substrates (Adam, 2007).

The main functions ascribed to the processive ATP-dependent proteases in organelles are i) to degrade excess subunits of proteins, ii) to eliminate photo-oxidatively damaged proteins, and iii) to exert protein quality control (Sakamoto, 2006). The involvement of FtsH and DegP in the repair cycle of PSII in chloroplasts under high light, by degradation of D1 protein, is well documented (Kato and Sakamoto, 2009). Mitochondrial Lon and FtsH proteases are involved in the biogenesis and maintenance of the oxidative phosphorylation system (Janska et al., 2010).

Relation of Expression of ATP-Dependent Protease Complexes to Function

The proteasome is essential for proper cellular function, removing proteins containing translation errors, improper processing and irreparable damage. It participates in protein quality control for both cytosolic and endoplasmic located proteins via the ER-associated degradation pathway, involving the assistance of ER chaperones and retrograde transport back to the cytoplasm (Kurepa and Smalle, 2008). Up to 30% of the translation products are rapidly removed by the proteasome under non-stress conditions (Smalle and Vierstra, 2004). Various pathway mutants are lethal or hypersensitive to stresses that damage or denature cell proteins (Smalle and Vierstra, 2004). In addition, the Ub-proteasome pathway controls hormonal signalling, including stress signal transduction, as well as cell metabolism, by removing short-lived regulatory proteins and enzymes that direct rate-limiting steps of metabolite pathways (Vierstra, 2009). These indispensable housekeeping functions and the great complexity of the proteasome favour its primarily constitutive expression and a role in acclimation to stress rather than of rapid stress induction.

Plant cells contain 26S and 20S proteasomes which mediate Ub-dependent and Ub-independent proteolysis, respectively. It is established that increased 26S biogenesis supports increased growth and increased tolerance to misfolded protein stresses (heat-shock stress for example). In contrast, increase in the relative abundance of 20S leads to decreased growth and better performance in oxidative stress conditions, since 20S degrades oxidatively modified proteins in an ATP-independent manner (Kurepa et al., 2009).

Chloroplast Clp protease is principally a constitutively expressed enzyme that degrades numerous stromal proteins. Among its putative substrates are enzymes involved in metabolic pathways such as photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis. Other putative substrates of Clp have been described that are involved in housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling processes (Stanne et al., 2009). Constitutive expression of ClpP proteins, without significant changes, has been reported under various stress conditions. It appears that plastid and mitochondrial proteolysis is regulated, not through regulation of ClpP gene expression, but rather through interaction of the core complex with ClpS1,2 and other chaperone-like molecules, as well as through substrate recognition mechanisms (Peltier et al., 2004). FtsH transcript levels in chloroplasts are highly responsive to strong light stress while temperature stresses have no effect on FtsH transcript abundance (Adam, 2007). Expression of mitochondrial ATP-dependent proteases is rather constant during their development, with some changes found in flowers and seeds (Janska et al., 2010). Expression of the Deg protease, which is not dependent on ATP, is increased under salt, light and temperature stresses (Sakamoto, 2006).

Involvement of ATP-Dependent Protease Complexes in the Response to Drought

Water deficit resulted in decreased total ATP-dependent proteolytic activity in wheat genotypes (Wisniewski and Zagdanska, 2001), while acclimation to drought prevented this decline. ATP-dependent protein degradation under drought was greater in the leaves of acclimated than of non-acclimated stressed plants (Zagdanska and Wisniewski, 1998; Wisniewski and Zagdanska, 2001). This activity is associated mainly with the proteasome in the cytosol and nucleus and the ATP-dependent proteases in chloroplasts and mitochondria.

ATP consumption for energy-dependent proteolysis amounts to about half that required for protein synthesis, and increases substantially under water deficit conditions (Zagdanska, 1995).

Regulation of proteasome activity includes adjustment of both total cellular proteolytic potential and target specificity. Changes in total proteasome activity can be caused by altered proteasome abundance or by posttranslational modification of subunits without affecting proteasome content. Changes in target specificity can be brought about by subunit modification and by the incorporation of subunit variants (Kurepa and Smalle, 2008). Plomion et al. (2006) reported induction by drought of members of the protein degradation machinery (i.e. 20S proteasome, polyubiquitin) in poplar trees, at the transcript level and the proteome level (26S protease regulatory subunit). Up-regulation of the 20S proteasome subunit was found in a proteomic study of drought-treated alfalfa plants (Aranjuelo et al., 2011). Wan et al. (2011) described up-regulation of a ubiquitin-conjugating enzyme gene AhUBC2 in dehydrated peanut plants; constitutive expression of this gene in Arabidopsis resulted in improved water-stress tolerance. The AtAIRP1 gene, encoding an E3 Ub ligase, was rapidly and significantly induced by ABA and by abiotic stresses including drought, low temperature, and high salinity. AtAIRP1-overexpressing transgenic plants were highly resistant to severe water stress (Ryu et al., 2010). It appears that the sumoylation system is highly responsive to environmental cues and plays a regulatory role in plant stress responses. Low and high temperatures, drought, salt, and oxidative stresses induce SUMO conjugation to protein substrates (Reed et al., 2010; Miura and Hassegava, 2010). Arabidopsis plants exposed to drought stress accumulate increased levels of sumoylated proteins by an ABA-independent pathway which is, in part, dependent on the transient increase of E3 SUMO ligase levels in response to drought (Catala et al., 2007).

Transcript analysis of ClpP/ClpR genes indicates constitutive expression in roots and leaves of A. thaliana with rather minor changes under stress and in senescence (Zheng et al., 2002; Sinvany-Villalobo et al., 2004). Short-term moderate and severe stresses (desiccation, high salt, cold, heat, oxidation, wounding and high light) all failed to elicit significant or rapid increases in any chloroplast Clp protein. However, increases in mRNA and protein content for ClpD and several ClpP isomers did occur during long-term high light and cold acclimation of Arabidopsis plants. These results reveal the great complexity of Clp proteins within the stroma of plant chloroplasts (Nakashima et al., 1997; Zheng et al., 2002). Immunoblotting analysis showed an enhancement of Clp proteases in drought treated wheat plants (Demirevska et al., 2008a).

Both FtsH genes in maize were constitutively expressed, the expression level of ZmFtsH2B transcripts being higher than that of ZmFtsH2A. Under polyethylene glycol, cold, high salt and ABA treatments, ZmFtsH2B transcription in leaves was markedly up-regulated, while ZmFtsH2A was constitutively expressed in both leaves and roots. However, drought tolerance of transgenic tobaccos overexpressing ZmFtsH2A and ZmFtsH2B was not greater than that of wild-type controls (Yue et al., 2010). Decreased abundance of FtsH was established in a susceptible Kentucky bluegrass cultivar under drought stress (Xu and Huang, 2010). Li et al. (2010) established that Arabidopsis Lon protease (atlon4) mutant is more sensitive to drought stress than wild-type plants.