Effect of stroma on the behavior of temoporfin-loaded lipid nanovesicles inside the stroma-rich head and neck carcinoma spheroids.

BACKGROUND: Despite the highly expected clinical application of nanoparticles (NPs), the translation of NPs from lab to the clinic has been relatively slow. Co-culture 3D spheroids account for the 3D arrangement of tumor cells and stromal components, e.g., cancer-associated fibroblasts (CAFs) and extracellular matrix, recapitulating microenvironment of head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated how the stroma-rich tumor microenvironment affects the uptake, penetration, and photodynamic efficiency of three lipid-based nanoformulations of approved in EU photosensitizer temoporfin (mTHPC): Foslip® (mTHPC in conventional liposomes), drug-in-cyclodextrin-in-liposomes (mTHPC-DCL) and extracellular vesicles (mTHPC-EVs). RESULTS: Collagen expression in co-culture stroma-rich 3D HNSCC spheroids correlates with the amount of CAFs (MeWo cells) in individual spheroid. The assessment of mTHPC loading demonstrated that Foslip®, mTHPC-DCL and mTHPC-EVs encapsulated 0.05 × 10- 15 g, 0.07 × 10- 15 g, and 1.3 × 10- 15 g of mTHPC per nanovesicle, respectively. The mid-penetration depth of mTHPC NPs in spheroids was 47.8 µm (Foslip®), 87.8 µm (mTHPC-DCL), and 49.7 µm (mTHPC-EVs), irrespective of the percentage of stromal components. The cellular uptake of Foslip® and mTHPC-DCL was significantly higher in stroma-rich co-culture spheroids and was increasing upon the addition of serum in the culture medium. Importantly, we observed no significant difference between PDT effect in monoculture and co-culture spheroids treated with lipid-based NPs. Overall, in all types of spheroids mTHPC-EVs demonstrated outstanding total cellular uptake and PDT efficiency comparable to other NPs. CONCLUSIONS: The stromal microenvironment strongly affects the uptake of NPs, while the penetration and PDT efficacy are less sensitive to the presence of stromal components. mTHPC-EVs outperform other lipid nanovesicles due to the extremely high loading capacity. The results of the present study enlarge our understanding of how stroma components affect the delivery of NPs into the tumors.

Binding of a Soluble meso-Tetraarylporphyrin to Human Galectin-7 Induces Oligomerization and Modulates Its Pro-Apoptotic Activity.

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.

An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates.

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.

Dissecting structural and electronic effects in inducible nitric oxide synthase.

Nitric oxide synthases (NOSs) are haem-thiolate enzymes that catalyse the conversion of L-arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide a hydrogen bond for oxygen activation (O-O scission). We present a study of native iNOS compared with iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to those of their native counterparts. Single turnover reactions catalysed by iNOSoxy with L-Arg (first reaction step) or N-hydroxy-L-arginine (second reaction step) showed that mesohaem substitution triggered higher rates of Fe(II)O2 conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared with the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency towards NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.

G-quadruplex conformation and dynamics are determined by loop length and sequence.

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.

Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

Molecular interactions and solubilization of structurally related meso-porphyrin photosensitizers by amphiphilic block copolymers (Pluronics).

The influence of four Pluronics block copolymers (i.e. F68, P123, F127, and L44) on the aggregation and solubilization of five structurally related meso-tetraphenyl porphyrin photosensitizers (PS) as model compounds for use in Photodynamic Therapy of cancer (PDT) was evaluated. Interactions between the PSs and Pluronics were studied at micromolar concentration by means of UV-Vis absorption spectrometry and by kinematic viscosity (υ) and osmolarity measurements at millimolar concentrations. Pluronic micelles were characterized by size and zeta potential (ζ) measurements. The morphology of selected PS-Pluronic assemblies was studied by atomic force microscopy (AFM). While hydrophobic 5,10,15,20-Tetrakis(4-hydroxyphenyl) porphine (THPP) seemed to be solubilized in the Pluronic micellar cores, amphiphilic di(monoethanolammonium) meso-tetraphenyl porphine disulphonate (TPPS2a) was likely bound to the micellar palisade layer. Hydrophilic PSs like 5,10,15,20-Tetrakis (4-trimethylaniliniumphenyl) porphine (TAPP) seemed to form complexes with Pluronic unimers and to be distributed among the micellar coronas. TPPS2a aggregated into a network which could be broken at Pluronic concentration [Formula: see text] cmc, but would reconstitute in the presence of tonicity adjusting agents, e.g. sodium chloride (NaCl) or glucose.

Inhibition of heme uptake in Pseudomonas aeruginosa by its hemophore (HasA(p)) bound to synthetic metal complexes.

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.

RNA and DNA complexes with hemin [Fe(III) heme] are efficient peroxidases and peroxygenases: how do they do it and what does it mean?

Guanine-rich RNAs and DNAs from chromosomal telomeres and elsewhere that fold into guanine quadruplexes (G-quadruplexes), are found to complex tightly with porphyrins such as N-methylmesoporphyrin IX (NMM) and hemin [Fe(III) heme]. By themselves, these DNAs and RNAs are found to be efficient catalysts for porphyrin metallation. When complexed with hemin, under physiological conditions, these nucleic acids display robust peroxidase (one-electron oxidation), as well as peroxygenase (two-electron oxidation, or oxygen transfer) activity. These surprising catalytic properties, that frequently match the catalytic performance of natural peroxidase and P450 monooxygenase enzymes, have been the subject of significant mechanistic analysis, as well as having found utility in a wide range of biosensing and other applications. This review summarizes recent insights into a surprising yet fundamental property of many RNAs and DNAs, a property with undoubted ramifications for cellular oxidative disease, de novo hemoenzyme design, and our understanding of the evolution of early biocatalytic systems.

Dissection of porphyrin-induced conformational dynamics in the heme biosynthesis enzyme ferrochelatase.

Human ferrochelatase (EC 4.99.1.1) catalyzes the insertion ferrous iron into protoporphyrin IX as the last step in heme biosynthesis, an essential process to most organisms given the vast intracellular functions of heme. Even with multiple ferrochelatase structures available, the exact mechanism for iron insertion into porphyrin is still a matter for debate. It is clear, however, that conformational dynamics are important for porphyrin substrate binding, initial chelation of iron, insertion of iron into the macrocycle, and release of protoheme IX. In this work we characterize conformational and dynamic changes in ferrochelatase associated with porphyrin binding using the substrate mesoporphyrin (MPIX) and backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS). In general, regions surrounding the active site become more ordered from direct or indirect interactions with the porphyrin. Our results indicate that the lower lip of the active site mouth is preorganized for efficient porphyrin binding, with little changes in backbone dynamics. The upper lip region has the most significant change in HDX behavior as it closes the active site. This movement excludes solvent from the porphyrin pocket, but leads to increased solvent access in other areas. A water lined path to the active site was observed, which may be the elusive iron channel with final insertion via the M76/R164/Y165 side of the porphyrin. These results provide a rigorous view of the ferrochelatase mechanism through the inclusion of dynamic information, reveal new structural areas for functional investigation, and offer new insight into a potential iron channel to the active site.

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells.

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.

ABCG2 transports and transfers heme to albumin through its large extracellular loop.

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 µm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 µm.

Development of pH sensitive 2-(diisopropylamino)ethyl methacrylate based nanoparticles for photodynamic therapy.

Photodynamic therapy is an effective treatment for tumors that involves the administration of light-activated photosensitizers. However, most photosensitizers are insoluble and non-specific. To target the acid environment of tumor sites, we synthesized three poly(ethylene glycol) methacrylate-co-2-(diisopropylamino)ethyl methacrylate (PEGMA-co-DPA) copolymers capable of self-assembly to form pH sensitive nanoparticles in an aqueous environment, as a means of encapsulating the water-insoluble photosensitizer, meso-tetra(hydroxyphenyl)chlorin (m-THPC). The critical aggregation pH of the PEGMA-co-DPA polymers was 5.8-6.6 and the critical aggregation concentration was 0.0045-0.0089 wt% at pH 7.4. Using solvent evaporation, m-THPC loaded nanoparticles were prepared with a high drug encapsulation efficiency (approximately 89%). Dynamic light scattering and transmission electron microscopy revealed the spherical shape and 132 nm diameter of the nanoparticles. The in vitro release rate of m-THPC at pH 5.0 was faster than at pH 7.0 (58% versus 10% m-THPC released within 48 h, respectively). The in vitro photodynamic therapy efficiency was tested with the HT-29 cell line. m-THPC loaded PEGMA-co-DPA nanoparticles exhibited obvious phototoxicity in HT-29 colon cancer cells after light irradiation. The results indicate that these pH sensitive nanoparticles are potential carriers for tumor targeting and photodynamic therapy.

Nanoparticle albumin-bound mTHPC for photodynamic therapy: Preparation and comprehensive characterization of a promising drug delivery system.

Nanoparticle albumin-bound (nab)-technology is an industrial applicable manufacturing method for the preparation of albumin-based drug carriers of poorly water-soluble drugs. In the present study the advantages of nanotechnology, albumin as an endogenous protein with the capability of high tumor enrichment and the selective light activation of the photosensitizer Temoporfin (mTHPC) were combined to a new delivery system for oncological use. The herewith provided well-established photodynamic therapy may enable a beneficial alternative for the treatment of solid tumors. In the present study a reproducible method for the preparation of stable mTHPC-albumin nanoparticles via nab-technology was established. The nanoparticles were physicochemically characterized with regard to particle size and size distribution and the impact of this preparation method on nanoparticle as well as mTHPC stability was investigated. Nanoparticles with improved colloidal stability over a broad pH range and in the presence of physiological NaCl concentrations were achieved in high yield. Due to high pressure homogenization a certain oxidative decay of mTHPC was observed. Cell culture experiments revealed an effective cellular uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1). After light-activation high cytotoxicity was shown for photosensitizer loaded nanoparticles enabling the application of the proposed formulation in photodynamic therapy.

Antibody-catalyzed porphyrin metallation.

An antibody elicited to a distorted N-methyl porphyrin catalyzed metal ion chelation by the planar porphyrin. At fixed Zn2+ and Cu2+ concentrations, the antibody-catalyzed reaction showed saturation kinetics with respect to the substrate mesoporphyrin IX (2) and was inhibited by the hapten, N-methylmesoporphyrin IX (1). The turnover number of 80 hour-1 for antibody-catalyzed metallation of 2 with Zn2+ compares with an estimated value of 800 hour-1 for ferrochelatase. The antibody also catalyzed the insertion of Co2+ and Mn2+ into 2, but it did not catalyze the metallation of protoporphyrin IX (3) or deuteroporphyrin IX (4). The antibody has high affinity for several metalloporphyrins, suggesting an approach to developing antibody-heme catalysts for redox or electron transfer reactions.

A dialysis-based in vitro drug release assay to study dynamics of the drug-protein transfer of temoporfin liposomes.

Today, a growing number of nanotherapeutics is utilized to deliver poorly soluble compounds using the intravenous route of administration. The drug release and the direct transfer of the active pharmaceutical ingredient to serum proteins plays an important role in bioavailability and accumulation of the drug at the target site. It is closely related to the formation of a protein corona as well as the plasma protein binding of the compound. In the present study, two in vitro drug release methods, the flow-through cell and the dispersion releaser technology, were evaluated with regards to their capability to measure a time-resolved profile of the serum protein binding. In this context, the photosensitizer temoporfin and temoporfin-loaded liposomes were tested. While in the fine capillaries of the flow-through cell a rapid agglomeration of proteins occurred, the dispersion releaser technology in combination with the four-step model enabled the measurement of the transfer of drugs from liposomes to proteins. In presence of 10% of fetal calf serum approximately 20% of the model compound temoporfin were bound to serum proteins within the first 3 h. At higher serum concentration this binding remained stable for approximately 10 h.

Investigation of Foscan interactions with plasma proteins.

The present study investigates the interaction of the second generation photosensitizer Foscan with plasma albumin and lipoproteins. Spectroscopic studies indicated the presence of monomeric and aggregated Foscan species upon addition to plasma protein solutions. Kinetics of Foscan disaggregation in albumin-enriched solutions were very sensitive to the protein concentration and incubation temperature. Kinetic analysis demonstrated that two types of Foscan aggregated species could be involved in disaggregation: dimers with a rate constant of k1 = (2.30+/-0.15) x 10(-3) s(-1) and higher aggregates with rate constants varying from (0.55+/-0.04) x 10(-3) s(-1) for the lowest to the (0.17+/-0.02) x 10(-3) s(-1) for the highest albumin concentration. Disaggregation considerably increased with the temperature rise from 15 degrees C to 37 degrees C. Compared to albumin, Foscan disaggregation kinetics in the presence of lipoproteins displayed poorer dependency on lipoprotein concentrations and smaller variations in disaggregation rate constants. Gel-filtration chromatography analysis of Foscan in albumin solutions demonstrated the presence of aggregated fraction of free, non-bound to protein Foscan and monomeric Foscan, bound to protein.

Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: implications for the catalytic reaction mechanism.

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.

Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme.

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (DeltaH=-97.1 kJ x mol(-1)), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 degrees C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process.

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition.

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM-G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.