The centrality of PBGD expression levels on ALA-PDT efficacy.

Successful 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is dependent on efficient porphyrin synthesis in the inflicted cancer tissue, which is regulated by several enzymes. Irradiation of the tumor excites the light-sensitive porphyrins and results in ROS production and cell death. In this study we investigated the effect of the expression levels of two main enzymes in heme biosynthesis, ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), on the capacity of K562 cells to undergo cell death following ALA-PDT. We manipulated PBGD and ALAD expression levels by shRNAs and PBGD overexpressing plasmid. PBGD down-regulation induced an elevation in ALAD activity, while overexpression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum. ALA-PDT efficacy was directly correlated to PpIX levels. Thus, only ALAD-silenced cells were not affected by ALA+ irradiation, while following PBGD silencing, the accumulated PpIX, though decreased, was sufficient for successful ALA-PDT. The alterations in ALAD activity level initiated by changes in PBGD expression indicates PBGD's central role in heme synthesis. This enables efficient ALA-PDT, even when PBGD is not fully active. Conversely, ALAD loss resulted in reduced PpIX synthesis and consequently failure in ALA-PDT, due to the absence of compensation mechanism for ALAD.

Regulation of heme synthesis and proteasomal activity by copper: possible implications for Wilson's disease.

Wilson's disease (Wd) is a genetic disorder resulting in Cu2+ accumulation, and is caused by mutations in the ATP7B gene, the copper transporter. In vivo studies show a correlation between Cu2+ accumulation and malfunction of the heme biosynthesis pathway. In this study, we describe multiple effects of Cu2+ accumulation on heme synthesis, which, in turn, affect proteasomal activity. Cu2+ toxicity was examined in two hepatocellular carcinoma cell lines, HepG2 and Hep3B, with Hep3B cells containing an integrated hepatitis B virus genome. Exposure of HepG2 and Hep3B cells to Cu2+ inhibited the enzymes PBGD and ALAD of the heme synthesis pathway and, in parallel, upregulated heme oxygenase-1 (HO-1). Proto-porphyrin IX (PpIX) and the heme pool were reduced as a result of these processes. PpIX synthesis was found to be lower in cells expressing the mutant ATP7B (P1134P), compared to those expressing the WT enzyme. Proteasomal activity was inhibited under Cu2+ treatment in HepG2 cells; however, Cu2+ induced marked proteosomal acceleration in Hep3B cells. Under these conditions, Ub-conjugated proteins were gradually accumulated, whereas treatment with bathocuproine disulfonic acid (BCS), a Cu2+ chelator, reversed this effect. In conclusion, our data suggest that copper downregulates the heme synthesis pathway in hepatocellular cells and further reduces it in the presence of mutated ATP7B.

Delta-aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro.

Recently, considerable interest has been given to photodynamic therapy of cancer using delta-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after delta-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with delta-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM delta-aminolaevulinic acid for 27 h accumulated 6.07 x 10(-16) mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm(-2) irradiation after a 3-h incubation with delta-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 x 10(-16) mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with delta-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the delta-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.

Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.

Allosteric inhibition of human lymphoblast and purified porphobilinogen deaminase by protoporphyrinogen and coproporphyrinogen. A possible mechanism for the acute attack of variegate porphyria.

Variegate porphyria (VP) is characterized by photocutaneous lesions and acute neuropsychiatric attacks. Decreased protoporphyrinogen oxidase activity results in accumulation of protoporphyrin (ogen) IX and coproporphyrin (ogen) III. During acute attacks delta-aminolevulinic acid and porphobilinogen also increase, suggesting that porphobilinogen deaminase (PBG-D) may be rate limiting. We have examined the effects of porphyrinogens accumulating in VP on PBG-D activity in Epstein-Barr virus-transformed lymphoblast sonicates from 12 VP and 12 control subjects. Protoporphyrinogen oxidase activity was decreased and protoporphyrin increased in VP lymphoblasts. PBG-D in control lymphoblasts obeyed Michaelis-Menten kinetics (Vmax 28.7 +/- 1.8 pmol/mg per h, Hill coefficient 0.83 +/- 0.07). VP sonicates yielded sigmoidal substrate-velocity curves that did not obey Michaelis-Menten kinetics. Vmax was decreased (21.2 +/- 2.0 pmol/mg per h) and the Hill coefficient was 1.78 +/- 0.17. Addition of protoporphyrinogen IX and coproporphyrinogen III to control sonicates yielded sigmoidal PBG-D substrate-velocity curves and decreased PBG-D Vmax. Addition of porphyrins or uroporphyrinogen III did not affect PBG-D activity. Removal of endogenous porphyrin (ogens) from VP sonicates restored normal PBG-D kinetics. Purified human erythrocyte PBG-D obeyed Michaelis-Menten kinetics (Vmax 249 +/- 36 nmol/mg per h, Km 8.9 +/- 1.5 microM, Hill coefficient 0.93 +/- 0.14). Addition of protoporphyrinogen yielded a sigmoidal curve with decreased Vmax. The Hill coefficient approached 4. These findings provide a rational explanation for the increased delta-aminolevulinic acid and porphobilinogen during acute attacks of VP.

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes.

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.

Acción fotodinámica y no-fotodinámica de las porfirinas sobre la aminolevulico dehidrasa en sangre normal y de pacientes con porfiria cutanea tardia (AU) / Photodinamic action and non-photodinamic action of the porphyrins in aminolevuleico dehydrase in normal blood, and patients with porphyria cutanea tarda

Se ha investigado la acción de concentraciones variables de uroporfirina I, uroporfirinógeno I y mezclas de porfirina aisladas de plasma y orina de pacientes porfíricos sobre la actividad de la alfa-aminolevúlico dehidrasa (ALA-D) de sangre de individuos normales y pacientes con PCT, en diferentes condiciones de iluminación, a 37-C y luego de 2 horas de exposición a la porfirina. La Uro I y el Urogen I inactivan la enzima en la oscuridad, efecto dependiente de la concentración que llega al 30-60% a valore de 10 µM del tetrapirrol. El Urogen I es un inhibidor más efectivo que la Uro I. La presencia de cantidades variables de mezclas de porfirinas aisladas del plasma y orina de pacientes con PCT, en la enzima de sangre normal y porfírica, produce también una inactivación independiente y una dependiente de la luz que aumenta a concentraciones crecientes de la mezcla, a partir de un valor umbral del orden de 1 - 1,5 µM por debajo del cual, los pigmentos no ejercen ningún tipo de inhibición

Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase.

The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA- mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free alpha-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino[5-14C]levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the alpha-substituted substrate analogue alpha-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cumulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH3 or H2O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

Comparative inhibition of hepatic hydroxymethylbilane synthase by both hard and soft metal cations.

The in vitro inhibition of hydroxymethylbilane synthase (EC 4.3.1.8, uroporphyrinogen I synthetase) obtained from livers of Sprague-Dawley rats has been studied with a wide range of di- and tri-valent metal ions. After purification by cell lysis, heat treatment, and centrifugation, the stable, soluble enzyme yielded sigmoidal inhibition curves with increasing concentrations of each of the 16 test ions. Using the negative logarithm of metal concentration for 50% inhibition (the pM50 value), the metal ions could be classified according to their Klopman hardness values. Very soft ions including Hg2+, intermediate ions including Cr3+, and very hard ions including Al3+ all yielded large pM50 values indicating strong inhibition. In comparison to known metal-ion chemical behaviour, these three ions could indicate three different types of inhibitory binding sites at or near the active site: Hg2+ corresponding to sulfur in cysteine, Cr3+ corresponding to nitrogen in histidine, and Al3+ corresponding to oxygen in carboxyl groups. The presence of the first two sites is also indicated by the pH dependence of activity.