SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

Five-mer peptides prevent short-term spatial memory deficits in Aß25-35-induced Alzheimer's model mouse by suppressing Aß25-35 aggregation and resolving its aggregate form.

BACKGROUND: The development of drugs for Alzheimer's disease (AD), which is related to the misfolding and aggregation of amyloid-ß (Aß), is high in demand due to the growing number of AD patients. In this study, we screened 22 kinds of 5-mer synthetic peptides derived from the Box A region of Tob1 protein to find a peptide effective against Aß aggregation. METHODS: A Thioflavin T (ThT) assay was performed to evaluate aggregation and screen aggregation inhibitors. Male ICR mice (6 weeks old) were administered saline, 9 nmol Aß25-35, or a mixture of 9 nmol Aß25-35 and 9 nmol GSGFK in the right lateral ventricle. Short-term spatial memory was assessed through Y-maze. Microglia cells (BV-)2 cells were plated on 24-well plates (4 × 104 cells/well) and incubated for 48 h, and then, the cells were treated with 0.01, 0.05, 0.1, 0.2, or 0.5 mM GSGFK. After incubation for 24 h, bead uptake was evaluated using a laser confocal microscope and Cytation 5. RESULTS: We found two kinds of peptides, GSGNR and GSGFK, that were not only suppressed by aggregation of Aß25-35 but also resolved the aggregated Aß25-35. Results obtained from the Y-maze test on an Aß25-35-induced AD model mouse indicated that GSGFK prevents the deficits in short-term memory induced by Aß25-35. The effect of GSGFK on phagocytosis in BV-2 cells proved that GSGFK activates the phagocytic ability of microglia. CONCLUSIONS: In conclusion, 5-mer peptides prevent short-term memory deficit in Aß25-35 induced AD model mouse by reducing the aggregated Aß25-35. They may also upregulate the phagocytic ability of microglia, which makes 5-mer peptides suitable candidates as therapeutic drugs against AD.

Structure-Activity Relationship of 5-mer Catalytides, GSGYR and RYGSG.

We recently discovered JAL-TA9 (YKGSGFRMI), a short hydrolytic peptide that we termed a Catalytide. The catalytic center of JAL-TA9 was modeled using MM2 and MMFF94 parameters and identified as GSGFR. Additionally, a structure-activity relationship study showed that GSGYR cleaved Aß11-29. Here, we developed a novel Catalytide in silico. Molecular dynamics simulations of GSGYR and RYGSG using MM2 and MMFF94 parameters suggested that both peptides may form catalytic triads and oxyanion holes. The hydrolytic potency of RYGSG was five times higher than that of GSGYR. Moreover, both peptides showed three common cleavage positions for Aß11-29; namely, L17-V18, V18-F19, and E22-D23. The aggregation ratio analyzed by the thioflavin-T assay correlated well with proteolytic activity, suggesting that the aggregation of Aß11-29 was suppressed by the cleavage reaction. Docking simulations with the carbonyl carbon of L17 or the carbonyl carbon of E22 in Aß11-29 were conducted using the secondary structures of GSGYR and RYGSG. The distance between the hydroxyl group of serine and the carbonyl carbon of the two cleavage sites proved that RYGSG was closer to Aß11-29 than to GSGYR. This study demonstrated that Catalytides are useful for understanding structure-activity relationships.

Direct Delivery of ANA-TA9, a Peptide Capable of Aß Hydrolysis, to the Brain by Intranasal Administration.

We have recently reported Catalytides (Catalytic peptides) JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMI), which are the first Catalytides found to cleave Aß42. Although the Catalytides must be delivered to the brain parenchyma to treat Alzheimer's disease, the blood-brain barrier (BBB) limits their entry into the brain from the systemic circulation. To avoid the BBB, the direct route from the nasal cavity to the brain was used in this study. The animal studies using rats and mice clarified that the plasma clearance of ANA-TA9 was more rapid than in vitro degradation in the plasma, whole blood, and the cerebrospinal fluid (CSF). The brain concentrations of ANA-TA9 were higher after nasal administration than those after intraperitoneal administration, despite a much lower plasma concentration after nasal administration, suggesting the direct delivery of ANA-TA9 to the brain from the nasal cavity. Similar findings were observed for its transport to CSF after nasal and intravenous administration. The concentration of ANA-TA9 in the olfactory bulb reached the peak at 5 min, whereas those in the frontal and occipital brains was 30 min, suggesting the sequential backward translocation of ANA-TA9 in the brain. In conclusion, ANA-TA9 was efficiently delivered to the brain by nasal application, as compared to other routes.

Amyloid beta cleavage by ANA-TA9, a synthetic peptide from the ANA/BTG3 Box A region.

INTRODUCTION: We recently discovered a short synthetic peptide derived from the ANA/BTG3 protein Box A region called ANA-TA9 (SKGQAYRMI), which possesses catalytic activity. Herein we demonstrated the proteolytic activity of ANA-TA9 against amyloid beta 42 (Aß42). METHODS: The proteolytic activity of ANA-TA9 against both the authentic soluble form Aß42 (a-Aß42) and the solid insoluble form Aß42 (s-Aß42) was analyzed by high-performance liquid chromatography and mass spectrometry. Plasma clearance, brain uptake, and cell viability were examined. RESULTS: ANA-TA9 cleaved not only a-Aß42 but also s-Aß42. Proteolytic activity was partially inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor. Plasma clearance was very rapid, and the brain concentration indicated efficient brain delivery of ANA-TA9 via nasal application. Cell viability analysis indicated that ANA-TA9 did not display toxicity. DISCUSSION: ANA-TA9 is an attractive potential candidate for the development of novel peptide drugs in Alzheimer's disease treatment.

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors.

BACKGROUND: Glial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. PURPOSE: Here, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity. METHODS: Engulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl2-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox. RESULTS: Although treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl2 was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl2. CONCLUSION: These findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.

Catalytides derived from the Box A region in the ANA/BTG3 protein cleave amyloid-ß fragment peptide.

We have recently reported about shorter proteolytic peptides termed Catalytide as general name. JAL-TA9 (YKGSGFRMI), a fragment peptide derived from Box A region of Tob1 protein, is the first Catalytide and cleaves Aß42 and its fragment peptides. Herein, we demonstrate the enzymatic properties of ANA-TA9 corresponding region to JAL-TA9 in ANA/BTG3 protein. ANA-TA9 showed the auto-proteolytic activity and cleaved 3 kinds of synthetic fragment peptides derived from Aß42, especially on the central region of Aß42 with a serine protease like activity. Interestingly, 2 kinds of components, ANA-SA5 (SKGQA) and ANA-YA4 (YRMI), also showed similar proteolytic activity. These results indicate that ANA-TA9 is composed of two different Catalytides.

Effects of Cu2+ on conformational change and aggregation of hPrP180-192 with a V180I mutation of the prion protein.

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNµ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a ß-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc.

The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aß11-29) under physiological conditions. The kcat of JAL-TA9 was 4.58 × 10-4 min-1 against MMP18-33 and 6.5 × 10-4 min-1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the kcat is 247.2 × 105 min-1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aß11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs.

Attenuation of zinc-enhanced inflammatory M1 phenotype of microglia by peridinin protects against short-term spatial-memory impairment following cerebral ischemia in mice.

Activated microglia exhibit two opposite activation states, the inflammatory M1 and the anti-inflammatory M2 activation states. In the mammalian brain, ischemia elicits a massive release of zinc from hippocampal neurons, and the extracellular zinc primes M1 microglia-by inducing reactive oxygen species (ROS) generation-to enhance their production of proinflammatory cytokines, which ultimately results in short-term spatial memory impairment. Here, we examined how peridinin, a carotenoid in dinoflagellates, affects the zinc-enhanced inflammatory M1 phenotype of microglia. Treatment of microglia with 30-300 ng/mL peridinin caused a dose-dependent attenuation of zinc-enhanced interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNFα) secretion when M1 activation was induced by lipopolysaccharide exposure. Moreover, peridinin inhibited the increase in ROS levels in zinc-treated microglia without directly interacting with zinc. Notably, when mice were administrated peridinin (20-200 ng/animal) intracerebroventricularly 5 min before cerebral ischemia-reperfusion, the peridinin treatment not only suppressed the increase in expression of IL-1ß, IL-6, TNFα, and the microglial M1 surface marker CD16/32, but also protected the mice against ischemia-induced short-term spatial-memory impairment. Our findings suggest that peridinin prevents extracellular zinc-enhanced proinflammatory cytokine secretion from M1 microglia by inhibiting the increase in microglial ROS levels, and that this anti-inflammatory effect of peridinin might result in protection against deficits in short-term spatial memory.

MMP-7 cleaves amyloid ß fragment peptides and copper ion inhibits the degradation.

The extracellular deposition of amyloid ß (Aß) is known to be the fundamental cause of Alzheimer's disease (AD). Aß1-42, generated by ß-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu2+ affects the physicochemical properties of Aß1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aß peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer's disease. In this study, we examined the degradation activity of MMP-7 against various Aß1-42's fragment peptides and the effect of Cu2+. Although Aß22-40 was degraded by MMP-7 regardless of Cu2+, Cu2+ inhibited the degradation of Aß1-19, Aß11-20, and Aß11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aß1-42, and that Aß1-42 acquired resistance against MMP-7 cleavage through Cu2+-binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aß1-42, which gives rise to the pathology of AD.

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.

(7S)-Kaitocephalin as a potent NMDA receptor selective ligand.

A structure-activity relationship (SAR) study of kaitocephalin (KCP), known to be a potent naturally occurring NMDA receptor ligand, was performed. This study led us to the discovery of (7S)-kaitocephalin as a highly selective NMDA receptor ligand. It displayed a 22-fold higher degree of selectivity for the NMDA receptor over KCP, though the binding affinity of (7S)-KCP [Ki = 29 nM] was 3.7-fold less potent than that of KCP [Ki = 7.8 nM].

Prion fragment peptides are digested with membrane type matrix metalloproteinases and acquire enzyme resistance through Cu²âº-binding.

Prions are the cause of neurodegenerative disease in humans and other mammals. The structural conversion of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc) is thought to relate to Cu2+ binding to histidine residues. In this study, we focused on the membrane-type matrix metalloproteinases (MT-MMPs) such as MT1-MMP and MT3-MMP, which are expressed in the brain as PrPC-degrading proteases. We synthesized 21 prion fragment peptides. Each purified peptide was individually incubated with recombinant MT1-MMP or MT3-MMP in the presence or absence of Cu2+ and the cleavage sites determined by LC-ESI-MS analysis. Recombinant MMP-7 and human serum (HS) were also tested as control. hPrP61-90, from the octapeptide-repeat region, was cleaved by HS but not by the MMPs tested here. On the other hand, hPrP92-168 from the central region was cleaved by MT1-MMP and MT3-MMP at various sites. These cleavages were inhibited by treatment with Cu2+. The C-terminal peptides had higher resistance than the central region. The data obtained from this study suggest that MT-MMPs expressed in the brain might possess PrPC-degrading activity.

Turnover of focal adhesions and cancer cell migration.

Cells are usually surrounded by the extracellular matrix (ECM), and adhesion of the cells to the ECM is a key step in their migration through tissues. Integrins are important receptors for the ECM and form structures called focal adhesions (FAs). Formation and disassembly of FAs are regulated dynamically during cell migration. Adhesion to the ECM has been studied mainly using cells cultured on an ECM-coated substratum, where the rate of cell migration is determined by the turnover of FAs. However, the molecular events underlying the disassembly of FAs are less well understood. We have recently identified both a new regulator of this disassembly process and its interaction partners. Here, we summarize our understanding of FA disassembly by focusing on the proteins implicated in this process.

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses.

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine(1), O-acetyl Serine(3)]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline(15)]-ß-MSH, together with [phosphoserine(15)]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

Metal-binding ability of human prion protein fragment peptides analyzed by column switch HPLC.

The structural conversion of the prion protein (PrP) from the normal cellular isoform (PrP(C)) to the posttranslationally modified form (PrP(Sc)) is thought to relate to Cu²âº binding to histidine (H) residues. Traditionally, the binding of metals to PrP has been investigated by monitoring the conformational conversion using circular dichroism (CD). In this study, the metal-binding ability of 21 synthetic peptides representing regions of human PrP(C) was investigated by column switch high-performance liquid chromatography (CS-HPLC). The CS-HPLC system is composed of a metal chelate affinity column and an octadecylsilica (ODS) reversed-phase column that together enable the identification of metal-binding regardless of conformational conversion. Synthetic peptides were designed with respect to the position of H residues as well as the secondary structure of human PrP (hPrP). The ability of the octapeptide (PHGGGWGQ)-repeating region (OP-repeat) to bind metals was analyzed by CS-HPLC and supported by CD analysis, and indicated that CS-HPLC is a reliable and useful method for measuring peptide metal-binding. Peptides from the middle region of hPrP showed a high affinity for Cu²âº, but binding to Zn²âº, Ni²âº, and Co²âº was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu²âº, Zn²âº, Ni²âº, and Co²âº than OP-repeat region peptides. Interestingly, hPrP193-230, which contained no H residues, also bound to Cu²âº, Zn²âº, Ni²âº, and Co²âº, indicating that this region is a novel metal-binding site in the C-terminal region of PrP(C). The CS-HPLC method described in this study is useful and convenient for assessing metal-binding affinity and characterizing metal-binding peptides or proteins.

ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain.

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, ß-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to ß-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

ZF21 is a new regulator of focal adhesion disassembly and a potential member of the spreading initiation center.

Adherent cells migrate on extracellular matrices (ECM) by repeated spreading and contraction of the cell body. Focal adhesions (FAs) play a major role in the adherence of cells to the ECM and in the generation of the cellular forces that maintain morphology and allow cells to move. FAs also mediate bidirectional transmembrane signals in conjunction with growth factor receptors and signaling molecules. Although the mechanisms that regulate cell migration are not yet fully understood, the regulation of the formation and turnover of FAs is a key factor determining the rate and direction of cell migration. We recently identified a component of FAs termed ZF21, which is a member of a family of proteins characterized by the presence of a conserved phosphoinositide-binding motif. ZF21 promotes dephosphorylation of FAK at Tyr ( 397) upon microtubule extension to FAs and thereby regulates the disassembly of FAs in a microtubules-dependent manner. To obtain further insight into the regulation of cell adhesion by ZF21, we analyzed proteins associating with ZF21 by proteomic analysis. We identified 45 proteins including FA-related proteins and multiple RNA binding proteins that have been shown recently to be components of the spreading initiation center (SIC). SICs are cell adherent structures that can be observed only in the early stages of cell spreading and have been implicated in regulating the rate of cell spreading. In this article, we report new ZF21-binding proteins identified by proteomic analysis and discuss the potential functions of ZF21 in regulating disassembly of FAs.

Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution.

To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH3Hg+) and accumulated more mercury from CH3Hg+-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH3Hg+ to Hg2+, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH3Hg+ and Hg2+ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg0) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.