Bharati Mitra

The ATP Hydrolytic Activity of Purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was ∼3 μmol/(mg·min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

From Bortezomib to other Inhibitors of the Proteasome and Beyond

The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade will also be discussed.

Escherichia coli Soft Metal Ion-translocating ATPases

Life may have first arisen in deep oceanic hydrothermal vents that were rich in metals such as arsenic, lead, copper, and zinc (1). Maintaining suitable intracellular concentrations of essential metals such as copper and zinc while excluding toxic metals such as arsenic, lead, and cadmium was one of the earliest challenges of the first cells. This ancient environmental challenge was a driving force for the evolution of mechanisms for metal ion homeostasis and detoxification. Even today toxic metals enter the ecosphere from geochemical sources. For example, parts of the Midwestern and Northeastern United States have arsenic concentrations that exceed 10 mg/liter, the provisional guideline of the World Health Organization for arsenic in drinking water, or 50 mg/liter, the present United States Environmental Protection Agency recommended maximum. Arsenic in the water supply in southern and western Bangladesh and the adjacent regions of India has triggered a health catastrophe. It is little wonder that in every organism examined there are transport systems that detoxify metal ions by catalyzing extrusion from the cytosol. In this review three families of Escherichia coli transport ATPases that catalyze uptake or confer resistance to ions of the transition metals copper and zinc, the heavy metals cadmium and lead, and the metalloids arsenic and antimony will be described. As a group these pumps will be designated soft metal ion-translocating ATPases or, for convenience, soft metal ATPases, because many ionic species of these elements are chemically soft Lewis acids, as opposed to the hard Lewis acids of Groups I and II elements such as Na and Ca. Hard Lewis acids bind to proteins through relatively weak ionic interactions with hard Lewis bases such as the carboxyl oxygens of glutamate or aspartate residues. In contrast, soft Lewis acids (or simply soft metal ions) form strong bonds with soft Lewis bases such as the thiolates of cysteine residues and the imidazolium nitrogens of histidine residues. These nearly covalent interactions with cysteines and histidines in proteins account for much of the biological properties and toxicity of soft metal ions.

A Zn(II)-translocating P-type ATPase from Proteus mirabilis

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.

Prostate tissue metal levels and prostate cancer recurrence in smokers.

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (Nu2009=u200925) had significantly higher Cd (median ppb, pu2009=u20090.03) and lower Zn (pu2009=u20090.002) in non-neoplastic tissue than never-smokers (Nu2009=u200921). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, pu2009=u20090.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, pu2009=u20090.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, pu2009=u20090.039 and adjacent non-neoplastic, pu2009=u20090.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.

Association of Metals and Proteasome Activity in Erythrocytes of Prostate Cancer Patients and Controls

Information is lacking on the effects toxic environmental metals may have on the 26S proteasome. The proteasome is a primary vehicle for selective degradation of damaged proteins in a cell and due to its role in cell proliferation, inhibition of the proteasome has become a target for cancer therapy. Metals are essential to the proteasomes normal function and have been used within proteasome-inhibiting complexes for cancer therapy. This study evaluated the association of erythrocyte metal levels and proteasome chymotrypsin-like (CT-like) activity in age- and race-matched prostate cancer cases (nu2009=u200961) and controls (nu2009=u200961). Erythrocyte metals were measured by inductively coupled plasma mass spectrometry (ICP-MS). CT-like activity was measured by proteasome activity assay using a fluorogenic peptide substrate. Among cases, significant correlations between individual toxic metals were observed (r(arsenic–cadmium)u2009=u20090.49, pu2009<u20090.001; r(arsenic–lead)u2009=u20090.26, pu2009=u20090.04, r(cadmium–lead) 0.53, pu2009<u20090.001), but there were no significant associations between metals and CT-like activity. In contrast, within controls there were no significant associations between metals, however, copper and lead levels were significantly associated with CT-like activity. The associations between copper and lead and proteasome activity (r(copper-CT-like)u2009=u2009−0.28, pu2009=u20090.002 ; r(lead-CT-like)u2009=u20090.23, pu2009=u20090.011) remained significant in multivariable models that included all of the metals. These findings suggest that biologically essential metals and toxic metals may affect proteasome activity in healthy controls and, further, show that prostate cancer cases and controls differ in associations between metals and proteasome activity in erythrocytes. More research on toxic metals and the proteasome in prostate cancer is warranted.

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

Zinc, Cadmium, and Lead Resistance and Homeostasis

Metals such as zinc are required for life but can be toxic in excess. Other metals such as cadmium and lead have almost no known biological function, and can lead to cell damage and death even at low concentrations. Interestingly, all three metals are often recognized by the same gene regulators and membrane transporters. Therefore, an examination of the inherent chemical properties of these three metal ions is essential in understanding the basis of metal specificity displayed by target proteins responsible for metal homeostasis and resistance. The relationship between the chemical properties of these metals and similarities in structural responses they may elicit are discussed. The core elements regulating uptake, efflux, and sequestration of these metals are described and interpreted both biologically and chemically. Additional mechanisms aiding cell survival, such as precipitation of metal salts on the cell surface, are also mentioned.

Case-only gene–environment interaction between ALAD tagSNPs and occupational lead exposure in prostate cancer

Black men have historically had higher blood lead levels than white men in the U.S. and have the highest incidence of prostate cancer in the world. Inorganic lead has been classified as a probable human carcinogen. Lead (Pb) inhibits delta‐aminolevulinic acid dehydratase (ALAD), a gene recently implicated in other genitourinary cancers. The ALAD enzyme is involved in the second step of heme biosynthesis and is an endogenous inhibitor of the 26S proteasome, a master system for protein degradation and a current target of cancer therapy.

Gene-environment interactions between JAZF1 and occupational and household lead exposure in prostate cancer among African American men.

PurposeA single nucleotide polymorphism, rs10486567, in JAZF1 has consistently been associated with increased risk of prostate cancer. The physical interaction of zinc finger proteins, such as JAZF1, with heavy metals may play a role in carcinogenesis. This study assessed potential gene–environment statistical interactions (G×E) between rs10486567 and heavy metals in prostate cancer.MethodsIn a case-only study of 228 African American prostate cancer cases, G×E between rs10486567 and sources of cadmium and lead (Pb) were assessed. Unconditional logistic regression was used to estimate interaction odds ratios (IORs), and generalized estimating equations were used for models containing nested data. Case–control validation of IORs was performed, using 82 controls frequency matched to cases on age–race.ResultsAmong cases, a potential G×E interaction was observed between rs10486567 CC genotype and living in a Census tract with a high proportion of housing built before 1950, a proxy for household Pb exposure, when compared to CT or TT carriers (OR 1.81; 95xa0% CI 1.04–3.16; pxa0=xa00.036). A stronger G×E interaction was observed when both housing and occupational Pb exposure were taken into account (OR 2.62; 95xa0% CI 1.03–6.68; pxa0=xa00.04). Case–control stratified analyses showed the odds of being a CC carrier were higher in cases compared to controls among men living in areas with older housing (OR 2.03; CI 0.99–4.19; pxa0=xa00.05) or having high occupational Pb exposure (OR 2.50; CI 1.01–6.18; pxa0=xa00.05).ConclusionsIn African American men, the association between JAZF1 rs10486567 and prostate cancer may be modified by exposure to heavy metals such as Pb.