Cadmium pyrithione suppresses tumor growth in vitro and in vivo through inhibition of proteasomal deubiquitinase.

The ubiquitin-proteasome system (UPS) is indispensable to the protein quality control in eukaryotic cells. Due to the remarkable clinical success of using proteasome inhibitors for clinical treatment of multiple myeloma, it is anticipated that targeting the UPS upstream of the proteasome step be an effective strategy for cancer therapy. Deubiquitinases (DUB) are proteases that remove ubiquitin from target proteins and therefore regulate multiple cellular processes including some signaling pathways altered in cancer cells. Thus, targeting DUB is a promising strategy for cancer drug discovery. Previously, we have reported that metal complexes, such as copper and gold complexes, can disrupt the UPS via suppressing the activity of 19S proteasome-associated DUBs and/or of the 20S proteasomes, thereby inducing cancer cell death. In this study, we found that cadmium pyrithione (CdPT) treatment led to remarkable accumulation of ubiquitinated proteins in cultured cancer cells and primary leukemia cells. CdPT potently inhibited the activity of proteasomal DUBs (USP14 and UCHL5), but slightly inhibited 20S proteasome activity. The anti-cancer activity of CdPT was associated with triggering apoptosis via caspase activation. Moreover, treatment with CdPT inhibited proteasome function and repressed tumor growth in animal xenograft models. Our results show that cadmium-containing complex CdPT may function as a novel proteasomal DUB inhibitor and suggest appealing prospects for cancer treatment.

Insights into zinc and cadmium biology in the nematode Caenorhabditis elegans.

Zinc is an essential metal that is involved in a wide range of biological processes, and aberrant zinc homeostasis is implicated in multiple human diseases. Cadmium is chemically similar to zinc, but it is a nonessential environmental pollutant. Because zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to maintain zinc levels in response to dietary fluctuations. The nematode Caenorhabditis elegans is emerging as a powerful model system to investigate zinc trafficking and homeostasis as well as cadmium toxicity. Here we review genetic and molecular studies that have combined to generate a picture of zinc homeostasis based on the transcriptional control of zinc transporters in intestinal cells. Furthermore, we summarize studies of cadmium toxicity that reveal intriguing parallels with zinc biology.

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death.

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.

Transcriptional regulation and expression network responding to cadmium stress in a Cd-tolerant perennial grass Poa Pratensis.

Kentucky bluegrass has good capability to absorb and accumulate cadmium (Cd) through developed root system, thus having potential phytoremediation function in Cd contaminated soils. Understanding the molecular mechanisms of Cd tolerance and accumulation in this species will be crucial to generating novel Cd-tolerance cultivars through genetic improvement, while it has not well documented yet. In the present study, comparative transcriptome analysis was performed for the seedlings of high Cd-tolerant genotype (M) and low Cd-tolerant genotype (R) under Cd stress. A total of 7022 up-regulated and 1033 down-regulated transcripts were identified in M genotype, whereas, only 850 up-regulated and 846 down-regulated transcripts were detected in R. Further transcriptional regulation analysis in M genotype showed that Dof, MADS25, BBR-BPC, B3, bZIP23 and MYB30 might be the hub transcription factors in response to Cd stress due to the orchestrated multiple functional genes associated with carbohydrate, lipid and secondary metabolism, as well as signal transduction. Differential expressed genes involved in auxin, ethylene, brassinosteroid and ABA signalling formed signal transduction cascades, which interacted with hub transcription factors, thereby finally orchestrated the expression of multiple genes associated with cell wall and membrane stability, cell elongation and Cd tolerance, including IAAs, ARFs, SnRK2, PP2C, PIFs, BES1/BZR1, CCR, CAD, FATB, fabF and HACD. Additionally, post-transcriptional modification of CIPKs, MAPKs, WAXs, UBCs, and E3 ubiquitin ligases were identified and also involved in plant signalling pathways and abiotic resistance. The study could contribute to our understanding the transcriptional regulation and complex internal network associated with Cd tolerance in Kentucky bluegrass.

Zinc and Cadmium in the Aetiology and Pathogenesis of Osteoarthritis and Rheumatoid Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are inflammatory articular conditions with different aetiology, but both result in joint damage. The nutritionally essential metal zinc (Zn2+) and the non-essential metal cadmium (Cd2+) have roles in these arthritic diseases as effectors of the immune system, inflammation, and metabolism. Despite both metal ions being redox-inert in biology, they affect the redox balance. It has been known for decades that zinc decreases in the blood of RA patients. It is largely unknown, however, whether this change is only a manifestation of an acute phase response in inflammation or relates to altered availability of zinc in tissues and consequently requires changes of zinc in the diet. As a cofactor in over 3000 human proteins and as a signaling ion, zinc affects many pathways relevant for arthritic disease. How it affects the diseases is not just a question of zinc status, but also an issue of mutations in the many proteins that maintain cellular zinc homoeostasis, such as zinc transporters of the ZIP (Zrt-/Irt-like protein) and ZnT families and metallothioneins, and the multiple pathways that change the expression of these proteins. Cadmium interferes with zinc's functions and there is increased uptake under zinc deficiency. Remarkably, cadmium exposure through inhalation is now recognized in the activation of macrophages to a pro-inflammatory state and suggested as a trigger of a specific form of nodular RA. Here, we discuss how these metal ions participate in the genetic, metabolic, and environmental factors that lead to joint destruction. We conclude that both metal ions should be monitored routinely in arthritic disease and that there is untapped potential for prognosis and treatment.

Mechanism of cadmium-mediated inhibition of Msh2-Msh6 function in DNA mismatch repair.

The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd(2+) ions per Msh2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd(2+) occurs via a nonspecific mechanism. Cd(2+) and Msh2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd(2+):Msh2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the peptide backbone as well. Our study also shows that cadmium inactivates several unrelated enzymes similarly, consistent with a nonspecific mechanism of inhibition. Targeting of a variety of proteins, including Msh2-Msh6, in this generic manner would explain the marked broad-spectrum impact of Cd(2+) on biological processes. We propose that the presence of multiple nonspecific Cd(2+) binding sites on proteins and their propensity to change conformation on interaction with Cd(2+) are critical determinants of the susceptibility of corresponding biological systems to cadmium toxicity.

Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination.

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd(2+) bonds. As Cd(2+) coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.

Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na(+)-Ca2+ exchange.

In cardiac myocytes, calcium influx through the calcium channel is the primary pathway for triggering calcium release. Recently it has been suggested that the calcium-induced calcium release mechanism can also be activated indirectly by the sodium current, which elevates the sodium concentration under the cell membrane, thereby favoring the entry of "trigger" calcium via the sodium-calcium exchanger. To test this hypothesis, sodium current was suppressed by reducing the external sodium concentration or applying tetrodotoxin. At potentials positive to -30 millivolts, calcium release was unaffected. A small calcium release at more negative potentials could be attributed to partial activation of calcium channels, because it was unaltered by replacement of sodium with lithium and was blocked by cadmium. Thus, sodium influx or its accumulation does not initiate calcium release. In addition, sodium-calcium exchange-related calcium release at potentials positive to +80 millivolts has slower kinetics than calcium channel-induced release. Therefore, only the calcium channel gates the fast release of calcium from the sarcoplasmic reticulum in the range of the action potential.

Improved Cd, Zn and Mn tolerance and reduced Cd accumulation in grains with wheat-based cell number regulator TaCNR2.

Soil microelement deficiency and heavy metal contamination affects plant growth and development, but improving trace element uptake and reducing heavy metal accumulation by genetic breeding can help alleviate this. Cell number regulator 2 (TaCNR2) from common wheat (Triticum aestivum) are similar to plant cadmium resistance proteins, involved with regulating heavy metal translocation. Our aim was to understand the effect of TaCNR2 on heavy metal tolerance and translocation. In this study, real-time quantitative PCR indicated TaCNR2 expression in the wheat seedlings increased under Cd, Zn and Mn treatment. Overexpression of TaCNR2 in Arabidopsis and rice enhanced its stress tolerance to Cd, Zn and Mn, and overexpression in rice improved Cd, Zn and Mn translocation from roots to shoots. The grain husks in overexpressed rice had higher Cd, Zn and Mn concentrations, but the brown rice accumulated less Cd but higher Mn than wild rice. The results showed that TaCNR2 can transport heavy metal ions. Thus, this study provides a novel gene resource for increasing nutrition uptake and reducing toxic metal accumulation in crops.

Stress resistance in long-lived mouse models.

Cellular stress resistance has been observed in a variety of long-lived mouse systems. The Ames and Snell dwarf mice show altered hormonal profiles (low levels of growth hormone/IGF-1 and of other hormones). These altered hormonal profiles lead to physiological changes in cells, leading to increased resistance to multiple forms of stress including UV light, oxidative stress, heat, and the heavy metal cadmium. The cells also show resistance to carcinogen and senescence-like growth arrest induced by ambient oxygen. Thus, cellular stress resistance may confer resistance to various diseases associated with stress insults. Stress resistance has also been observed in various long-lived mice (hemizygous knockout of igf-1r, a mutation in p66(shc), and klotho overexpression) and in vitro CR (Carolie Restriction) system. Many of the long-lived mouse systems show reduction or inhibition of the insulin/IGF-1-FOXO pathway, thus suggesting that there may be an overlapping mechanism for increased life span. The insulin/IGF-1-FOXO pathway interlocks to several signal transduction pathways through AKT, FOXO, JNK, and other components. Taken together, stress resistance may be an essential function in cells that leads to increased longevity. I will summarize molecular basis of stress resistance and further discuss stress resistance in other systems.

Gene- and cell-type-specific effects of signal transduction cascades on metal-regulated gene transcription appear to be independent of changes in the phosphorylation of metal-response-element-binding transcription factor-1.

Post-translational modification of MTF-1 (metal-response-element-binding transcription factor-1) was suggested to play a role in its metalloregulatory functions. In the present study, pulse labelling and two-dimensional electrophoresis-Western blotting were used to demonstrate that, although MTF-1 is highly modified in vivo, its phosphorylation level does not rapidly change in response to metals, nor does its overall modification pattern. Recombinant MTF-1 was found to serve as an in vitro substrate for casein kinase II, c-Jun N-terminal kinase and protein kinase C, but inhibition of these kinases in vivo did not significantly change the modification pattern of MTF-1. Northern blotting revealed that inhibitors of casein kinase II and c-Jun N-terminal kinase severely attenuate the metal-induced transcription of the native chromatin-packaged metallothionein-I and zinc transporter-1 genes, whereas protein kinase C inhibitors exerted gene- and cell-type-specific effects on the metal regulation and basal expression of these two genes. A chromatin immunoprecipitation assay was used to demonstrate that none of these inhibitors prevent the metal-dependent recruitment of MTF-1 to the MT-I promoter. In brief, results of the present study suggest that protein kinases may not alter the phosphorylation state of MTF-1 during the rapid-response phase to metals, nor do they regulate the metal-dependent formation of a stable MTF-1-chromatin complex. Instead, protein kinases may exert their interdependent effects on metal-induced gene expression by acting on cofactors that interact with MTF-1.

Role of cadmium in hypertensive patients with renal parenchymal disease.

In our study we investigated 36 out-patients with renal disease; 22 of them were hypertensive. In all patients proteinuria was present (4.30 +/- 0.82 g protein/d) and renal involvement has been proved by renal biopsy. Blood cadmium in nonsmokers was significantly (P less than .05) lower than in smokers. Patients with renal hypertension showed a significantly higher (P less than .05) urine cadmium excretion/d (1.60 +/- 0.23 micrograms/d) compared to normotensives with a disease of the kidney (1.14 +/- 0.38 micrograms/d). Our results indicate that cadmium may be involved in the development of hypertension in patients with renal disease.

A potential role for cadmium in the etiology of varicocele-associated infertility.

OBJECTIVE: To determine whether mannose ligand receptor and acrosome reaction deficits in sperm from men with varicocele are related to the transition metal content of their semen. DESIGN: Cadmium and zinc in semen and blood plasma were assayed for fertile males, men without varicocele who required intracytoplasmic sperm injection to achieve fertilization, and men evaluated for potential varicocele-associated infertility. The relationship between actin cytoskeletal distributions and acrosome status was determined for fertile donor sperm in the presence and absence of exogenous cadmium. SETTING: University hospital-based molecular biology research laboratory. PATIENT(S): Patients from two university hospital-based IVF-assisted reproductive technology programs and two male urology private practices. INTERVENTION(S): Fertile donor sperm were exposed to exogenous cadmium during capacitating incubations followed by culture at temperatures up to 41 degrees C. MAIN OUTCOME MEASURE(S): Metal ion levels in semen and blood plasma were determined by graphite furnace atomic absorption spectroscopy. Motile sperm were examined for mannose ligand binding and the ability to undergo spontaneous and induced acrosome reactions. Unfixed, Triton-permeabilized sperm were probed with antiactin and antimyosin antibodies. RESULT(S): Cadmium was elevated and zinc was decreased in the seminal plasma of men with varicocele. The content of these metals in semen and blood was not correlated. Cadmium exposure in vitro reduced mannose receptor expression, acrosome exocytosis, and cytoskeletal formation by fertile donor sperm. CONCLUSION(S): Defects in transition metal regulation or excessive cadmium exposure are involved in varicocele-associated infertility.

Complex study of the physiological role of cadmium. III. Cadmium loading trials on broiler chickens.

Cadmium (Cd) loading trials were conducted on a total of 110 (3 x 10 and 4 x 20) broiler chickens prereared for 21 days. The control chickens received no cadmium, while chickens in the six treatment groups were given different doses of Cd as an aqueous solution of CdSO4 administered either into the crop or mixed in the feed. The chickens were kept in a climatized animal house and treated usually for 3-5 weeks (maximum 68 days), with the exception of group Cd-75 chickens which were treated up to 239 days of age. The chickens' health status, body mass and feed consumption were monitored throughout the trial. On days 14-20 and on day 42 of the trial 2 chickens per group, then at the end of trial a total of 25 chickens were killed in anaesthesia. These birds, together with chickens that died or were killed during the trial, were subjected to detailed gross pathological examination. From 11 organs (kidney, liver, spleen, testicle, brain, myocardium, skeletal muscle, lungs, digestive tract, pancreas, tubular bones) of these chickens samples were taken for assay for a total of 16 elements, as well as for light and electron microscopic examination. With the exception of groups Cd-30 and Cd-600, no abnormal clinical signs were observed in the first two weeks of the trial. Chickens of group Cd-30 died before day 8-12 of the trial among signs of complete anorexia, rapid emaciation, huddling and diarrhoea, while chickens of group Cd-600 died before day 28, showing similar clinical signs. The body mass of chickens fed a Cd-supplemented diet either remained constant or decreased substantially, in a degree proportional to the Cd load. The only exception was group Cd-2.5, in which the average body mass of birds at the end of week 8 slightly exceeded that of the controls. Four out of the 10 cockerel chicks fed a diet containing 75 ppm Cd up to 239 days of age died of intercurrent diseases; the remaining six grew well and reached a body mass of 3.8-4.3 kg. Feed conversion efficiency was satisfactory in the control group and in group Cd-2.5 (2.1 and 2.4 kg, respectively) and could not be evaluated in a realistic manner in the other groups. At necropsy, the cockerel chicks of groups Cd-30 and Cd-600 showed severe emaciation, liver and kidney degeneration, myocardial hypertrophy and cardiac dilatation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cadmium induces nuclear translocation of beta-catenin and increases expression of c-myc and Abcb1a in kidney proximal tubule cells.

Cadmium (Cd2+) induces renal proximal tubular (PT) damage, including disruption of the E-cadherin/beta-catenin complex of adherens junctions (AJs) and apoptosis. Yet, chronic Cd2+ exposure causes malignant transformation of renal cells. Previously, we have demonstrated that Cd(2+)-mediated up-regulation of the multidrug transporter Abcb1 causes apoptosis resistance in PT cells. We hypothesized that Cd2+ activates adaptive signaling mechanisms mediated by beta-catenin to evade apoptosis and increase proliferation. Here we show that 50 microM Cd2+, which induces cell death via apoptosis and necrosis, also causes a decrease of the trans-epithelial resistance of confluent WKPT-0293 Cl.2 cells, a rat renal PT cell model, within 45 min of Cd2+ exposure, as measured by electric cell-substrate impedance sensing. Immunofluorescence microscopy demonstrates Cd(2+)-induced decrease of E-cadherin at AJs and redistribution of beta-catenin from the E-cadherin/beta-catenin complex of AJs to cytosol and nuclei after 3 h. Immunoblotting confirms Cd(2+)-induced decrease of E-cadherin expression and translocation of beta-catenin to cytosol and nuclei of PT cells. RT-PCR shows Cd(2+)-induced increase of expression of c-myc and of the isoform Abcb1a at 3 h. The data prove for the first time that Cd2+ induces nuclear translocation of beta-catenin in PT cells. We speculate that Cd2+ activates beta-catenin/T-cell factor signaling to trans-activate proliferation and apoptosis resistance genes and promote carcinogenesis of PT cells.

Heavy metal stress and sulfate uptake in maize roots.

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.

Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase.

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.