Francesca Gullotta

CO metabolism, sensing, and signaling

CO is a colorless and odorless gas produced by the incomplete combustion of hydrocarbons, both of natural and anthropogenic origin. Several microorganisms, including aerobic and anaerobic bacteria and anaerobic archaea, use exogenous CO as a source of carbon and energy for growth. On the other hand, eukaryotic organisms use endogenous CO, produced during heme degradation, as a neurotransmitter and as a signal molecule. CO sensors act as signal transducers by coupling a “regulatory” heme‐binding domain to a “functional” signal transmitter. Although high CO concentrations inhibit generally heme‐protein actions, low CO levels can influence several signaling pathways, including those regulated by soluble guanylate cyclase and/or mitogen‐activated protein kinases. This review summarizes recent insights into CO metabolism, sensing, and signaling.

Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme-albumin.

The ibuprofen primary binding site FA3–FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1–Glu382 truncated HSA (tHSA)–heme‐Fe(III) and nitrosylated tHSA–heme‐Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA–heme‐Fe(III)‐mediated peroxynitrite isomerization and nitrosylated tHSA–heme‐Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA–heme and HSA–heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3–FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand‐binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.

Carbon monoxide: An unusual drug

The highly toxic gas carbon monoxide (CO) displays many physiological roles in several organs and tissues. Although many diseases, including cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration, and sleep disorders, have been linked to abnormal endogenous CO metabolism and functions, CO administration has therapeutic potential in inflammation, sepsis, lung injury, cardiovascular diseases, transplantation, and cancer. Here, insights into the CO‐based therapy, characterized by the induction or gene transfer of heme oxygenase‐1 and either gas or CO‐releasing molecule administration, are reviewed. 2012 IUBMB IUBMB Life, 2012

Deletion 2q37: An Identifiable Clinical Syndrome With Mental Retardation and Autism

Terminal deletion of the long arm of chromosome 2 is a rare chromosomal disorder characterized by low birth weight, delayed somatic and mental development, craniofacial defects, short neck, heart and lung congenital defects, and autistic features. We report on a girl with 46,XX.ish del(2)(q37.1) de novo karyotype, mental retardation, dysmorphic features, gastrointestinal anomalies, and autistic traits and compare her clinical manifestations with patients with the same deletion previously described in literature.

Drug binding to Sudlow's site I impairs allosterically human serum heme‐albumin‐catalyzed peroxynitrite detoxification

Heme endows human serum albumin (HSA) with globin‐like reactivity and spectroscopic properties. Here, the effect of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitrite isomerization to NO  3− by ferric HSA‐heme (HSA‐heme‐Fe(III)) is reported. Drugs binding to Sudlows site I impair dose‐dependently peroxynitrite isomerization by HSA‐heme‐Fe(III). The allosteric modulation of HSA‐heme‐Fe(III)‐mediated peroxynitrite isomerization by drugs has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlows site I or protrudes into the heme cleft (i.e., the fatty acid site 1, FA1), depending on ligand occupancy of either sites.

Reductive nitrosylation of ferric human serum heme-albumin.

Heme endows human serum albumin (HSA) with heme‐protein‐like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme‐albumin [HSA‐heme‐Fe(III)] are reported. All data were obtained at 20 °C. At pH 5.5, HSA‐heme‐Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA‐heme‐Fe(III) [HSA‐heme‐Fe(III)‐NO]. By contrast, at pH ≥ 6.5, the addition of NO to HSA‐heme‐Fe(III) leads to the transient formation of HSA‐heme‐Fe(III)‐NO in equilibrium with HSA‐heme‐Fe(II)‐NO+. Then, HSA‐heme‐Fe(II)‐NO+ undergoes nucleophilic attack by OH− to yield ferrous human serum heme‐albumin [HSA‐heme‐Fe(II)]. HSA‐heme‐Fe(II) further reacts with NO to give nitrosylated HSA‐heme‐Fe(II) [HSA‐heme‐Fe(II)‐NO]. The rate‐limiting step for reductive nitrosylation of HSA‐heme‐Fe(III) is represented by the OH−‐mediated reduction of HSA‐heme‐Fe(II)‐NO+ to HSA‐heme‐Fe(II). The value of the second‐order rate constant for OH−‐mediated reduction of HSA‐heme‐Fe(II)‐NO+ to HSA‐heme‐Fe(II) is 4.4 × 103 m−1·s−1. The present results highlight the role of HSA‐heme‐Fe in scavenging reactive nitrogen species.

Targeting the DNA Double Strand Breaks Repair for Cancer Therapy

Among several types of DNA lesions, the DNA double strand breaks (DSBs) are one of the most deleterious and harmful. Mammalian cells mount a coordinated response to DSBs with the aim of appropriately repair the DNA damage. Indeed, failure of the DNA damage response (DDR) can lead to the development of cancer-prone genetic diseases. The identification and development of drugs targeting proteins involved in the DDR is even more investigated, as it gives the possibility to specifically target cancer cells. Indeed, the administration of DNA repair inhibitors could be combined with chemo- and radiotherapy, thus improving the eradication of tumor cells. Here, we provide an overview about DSBs damage response, focusing on the role of the DSBs repair mechanisms, of chromatin modifications, and of the cancer susceptibility gene BRCA1 which plays a multifunctional role in controlling genome integrity. Moreover, the most investigated DSBs enzyme inhibitors tested as potential therapeutic agents for anti-cancer therapy are reported.

Determination of antituberculosis drug concentration in human plasma by MALDI‐TOF/TOF

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix‐assisted laser desorption/ionization source equipped with tandem time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti‐TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis‐infected patients is reported. The volume of the plasma sample was 200 μL. Plasma samples were cleaned‐up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 μL of 50% methanol‐0.03% formic acid solution (v/v), spiked with known amounts of anti‐TB drugs, mixed (1:1) with a saturated matrix solution (4‐hydroxybenzoic acid in 50% acetonitrile‐0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI‐TOF/TOF sample target plate. The anti‐TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti‐TB drug concentration range explored (the final anti‐TB drug concentration ranged from 0.20 to 200 pmol/μL). The absolute recovery of the anti‐TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI‐TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/μL, respectively.

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k(off) value, representing the rate-limiting step, can be directly determined. The k(off) value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k(on)) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k(off)=k(on)xK).

O2-mediated oxidation of ferrous nitrosylated human serum heme–albumin is limited by nitrogen monoxide dissociation

Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O(2)-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O(2)-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k=9.8 × 10(-5) and 8.3 × 10(-4) s(-1), and h=1.3 × 10(-4) and 8.5 × 10(-4) s(-1), in the absence and presence of rifampicin, respectively, at pH=7.0 and T=20.0 °C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O(2)-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O(2) does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O(2)-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.