Over-expression,purification and functional characterization of <Emphasis Type="Italic">Celosia</Emphasis> ClpS as a fused protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A ClpS homologue from Celosia cristata was expressed as maltose-binding fusion protein under the control of strong inducible tac promoter of pMALc2X vector in TB1 strain of Escherichia coli. SDS-PAGE analysis showed that fused ClpS is produced as about 63 kDa protein in recombinant bacteria. Expressed product was purified to homogeneity with a yield of about 31 mg/l of bacterial culture. The results indicated that heterologous expression of Celosia ClpS does not affect bacterial growth under different induced conditions. Total cellular antioxidant assessment results revealed that the induction of ClpS activates the bacterial antioxidative system. Since, the purified ClpS did not exhibit antioxidant activity in vitro, we speculated a functional corelation between bacterial proteolytic apparatus and its anti-oxidative system. This prediction may contribute to our better understanding of functional relationship between proteolytic and antioxidative systems in biological worlds in the future investigations.     

Molecular cloning of an actinobacterial-type ClpS gene from Celosia cristata expression library

In proteobacterial cytosol, ClpS protein is known as a molecular adaptor for substrate selectivity and proteolytic activity of the ATP-dependent chaperone-protease complex, ClpAP. ClpA-related ClpS is a small protein usually encoded immediately upstream of ClpA in the genome of proteobacteria. Recent bioinformatics analysis has revealed the presence of cyanobacterial-type ClpS or ClpC-related ClpS in organisms lacking ClpA, including all the plant species sequenced to date. Here we report the identification of an actinobacterial homologue of the ClpS (possibly Clp-related) gene from a plant system. A cDNA, spanning 566 bp with a complete coding region corresponding to 132 amino acids, was isolated from a Celosia cristata expression library constructed on a λ TriplEX2 vector. This cDNA product was considered to be an ATP-dependent Clp protease adaptor and was designated as Celosia actinobacterial-type ClpS, since it contains a highly conserved domain belonging to the ClpS family of proteins from actinobacteria. Celosia ClpS is about 80% identical to actinobacterial ClpS proteins in its overall deduced amino acid sequence. Based on this finding, we may define a novel target of ATP-dependent Clp complex in a plant system or speculate the presence of a second type of molecular chaperone besides ClpC in plants, as predicted for actinobacteria.     

ClpS1 Is a Conserved Substrate Selector for the Chloroplast Clp Protease System in Arabidopsis

Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.     

Functional properties of dioscorin, a soluble viscous protein from Japanese yam (Dioscorea opposita thunb.) tuber mucilage Tororo

A soluble viscous protein was purified from yam (Dioscorea opposita Thunb.) tuber mucilage tororo by chromatographic steps, and its functional properties were estimated. The purified dioscorin having the molecular weight of about 200 kDa exhibited high scavenging activities against hydroxyl radicals (IC50 = 195.1 microg/ml) and superoxide anion radicals (IC50 = 92.7 microg/ml). Moreover, it showed extremely high angiotensin I-converting enzyme inhibitory activity (IC50 = 41.1 microg/ml). The results suggested that yam D. opposita tuber has a wide spectrum of strong antioxidative and antihypertensive activities and it could be utilized as a source of natural antioxidant.     

Antioxidant Activity of Antiviral Proteins from Celosia cristata

The antioxidant activity test of Celosia cristata antiviral proteins (CCP-25 and CCP-27) using ferric reducing antioxidant power (FRAP) assay in vitro indicated that these proteins are strong antioxidants. The increase in activities of redox-enzymes such as peroxidase, catalase and polyphenol oxidase on tobacco mosaic virus (TMV) inoculation of test plants, was inhibited when plants were treated with CCP-25 before TMV inoculation. The activity of phenylalanine ammonia lyase, involved in biosynthesis of antioxidative compounds was also inhibited. This is the first ever report where plant antiviral proteins have been shown to have strong antioxidative property. A possible correlation between antioxidant activity of CCPs and their antiviral activity is speculated.     

ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli : Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

Discovery of a Unique Clp Component,ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.     

Bioinformatic analysis of ClpS,a protein module involved in prokaryotic and eukaryotic protein degradation

ClpS is a small protein, usually encoded immediately upstream of ClpA in the genomes of proteobacteria. Recent results show that it is a molecular adaptor for substrate recognition by ClpA in Escherichia coli. We analyzed ClpS by bioinformatic methods and found that ClpS homologs are also found in organisms that lack ClpA, such as actinobacteria, cyanobacteria, and plant chloroplasts. Furthermore, ClpS is homologous to a domain in the eukaryotic E3 ubiquitin ligase, N-recognin. This domain has previously been described as responsible for the recognition of type 2 N-end rule substrates. Despite very low levels of sequence similarity to proteins of known structure, there appears to be substantial structural similarity between ClpS and the C-terminal domain of ribosomal protein L7/12 (1CTF).     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.     

Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from <Emphasis Type="Italic">Celosia cristata</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.     

Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin

Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (-44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.     

Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH

Purified serum paraoxonase (PON1) had been shown to attenuate the oxidation of LDL in vitro. We critically reevaluated the antioxidant properties of serum PON1 in the in vitro assays initiated with copper or the free radical generator 2,2'-azobis-2-amidinopropane hydrochloride (AAPH). The antioxidant activity of different purified PON1 preparations did not correlate with their arylesterase (AE), lactonase, or phospholipase A2 activities or with the amounts of detergent or protein. Dialysis of three of these preparations resulted in a 30-40% loss of their AE activities but in a complete loss of their antioxidant activities. We also followed the distribution of the antioxidant activity during human serum PON1 purification by two purification methods. The antioxidant activity of the anion-exchange chromatography fractions did not copurify with PON1 using either method and could largely be accounted for by the "antioxidant" activity of the detergent present. In conclusion, using the copper or AAPH in vitro assays, no PON1-mediated antioxidant activity was detected, suggesting that the removal of PON1 from its natural environment may impair its antioxidative activity and that this assay with highly purified PON1 may be an inappropriate method with which to study the antioxidative properties of the enzyme.     

ClpS, a substrate modulator of the ClpAP machine

In the bacterial cytosol, ATP-dependent protein degradation is performed by several different chaperone-protease pairs, including ClpAP. The mechanism by which these machines specifically recognize substrates remains unclear. Here, we report the identification of a ClpA cofactor from Escherichia coli, ClpS, which directly influences the ClpAP machine by binding to the N-terminal domain of the chaperone ClpA. The degradation of ClpAP substrates, both SsrA-tagged proteins and ClpA itself, is specifically inhibited by ClpS. In contrast, ClpS enhanced ClpA recognition of two heat-aggregated proteins in vitro and, consequently, the ClpAP-mediated disaggregation and degradation of these substrates. We conclude that ClpS modifies ClpA substrate specificity, potentially redirecting degradation by ClpAP toward aggregated proteins.     

A single amino acid substitution alters ClpS2 binding specificity

ClpS2 is a small protein under development as a probe for selectively recognizing N-terminal amino acids of N-degron peptide fragments. To understand the structural basis of ClpS2 specificity for an N-terminal amino acid, all atom molecular dynamics (MD) simulations were conducted using the sequence of a bench-stable mutant of ClpS2, called PROSS. We predicted that a single amino acid leucine to asparagine substitution would switch the specificity of PROSS ClpS2 to an N-terminal tyrosine over the preferred phenylalanine. Experimental validation of the mutant using a fluorescent yeast-display assay showed an increase in tyrosine binding over phenylalanine, in support of the proposed hypothesis.     

DUF538 Protein Super Family is Predicted to be the Potential Homologue of Bactericidal/Permeability-Increasing Protein in Plant System

DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.     

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.     

Crystal structure of the heterodimeric complex of the adaptor,ClpS, with the N-domain of the AAA+ chaperone,ClpA

Substrate selectivity and proteolytic activity for the E. coli ATP-dependent protease, ClpAP, is modulated by an adaptor protein, ClpS. ClpS binds to ClpA, the regulatory component of the ClpAP complex. We report the crystal structure of ClpS in complex with the isolated N-terminal domain of ClpA in two different crystal forms at 2.3- and 3.3-A resolution. The ClpS structure forms an alpha/beta-sandwich and is topologically analogous to the C-terminal domain of the ribosomal protein L7/L12. ClpS contacts two surfaces on the N-terminal domain in both crystal forms; the more extensive interface was shown to be favored in solution by protease protection experiments. The N-terminal 20 residues of ClpS are not visible in the crystal structures; the removal of the first 17 residues produces ClpSDeltaN, which binds to the ClpA N-domain but no longer inhibits ClpA activity. A zinc binding site involving two His and one Glu residue was identified crystallographically in the N-terminal domain of ClpA. In a model of ClpS bound to hexameric ClpA, ClpS is oriented with its N terminus directed toward the distal surface of ClpA, suggesting that the N-terminal region of ClpS may affect productive substrate interactions at the apical surface or substrate entry into the ClpA translocation channel.     

定量蛋白质组学分析ClpS在分枝杆菌耐药中的功能

ClpS是原核生物蛋白质降解复合物ClpAPS的重要组成成分,它可以识别某些特定的氨基酸序列并将其呈递给ClpAP以促进其降解。同时,ClpS也抑制了其他蛋白质底物的降解。本研究通过在耻垢分枝杆菌中过度表达ClpS,发现所构建的重组菌株提高了利福平的抗药性。应用定量蛋白质组学技术,我们系统地分析了过度表达ClpS对于细菌蛋白质组的影响,并推测出细菌抗利福平的分子机制:ClpS促进稳态的调整、促进药物沉降以及加速药物代谢。本研究首次通过改变细菌降解复合物的相关蛋白的表达增加细菌的抗药性,并证明蛋白质组学技术是细菌的抗药性研究以及耐药株筛选的重要工具。     

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.