Alessandra di Masi

Human serum albumin: From bench to bedside

Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. HSA displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Indeed, HSA represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays (pseudo-)enzymatic properties. HSA is a valuable biomarker of many diseases, including cancer, rheumatoid arthritis, ischemia, post-menopausal obesity, severe acute graft-versus-host disease, and diseases that need monitoring of the glycemic control. Moreover, HSA is widely used clinically to treat several diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutrition support, resuscitation, and hypoalbuminemia. Recently, biotechnological applications of HSA, including implantable biomaterials, surgical adhesives and sealants, biochromatography, ligand trapping, and fusion proteins, have been reported. Here, genetic, biochemical, biomedical, and biotechnological aspects of HSA are reviewed.

Retinoic acid receptors: From molecular mechanisms to cancer therapy

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

NBS1 Heterozygosity and Cancer Risk.

Biallelic mutations in the NBS1 gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBS1 gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBS1 heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBS1 gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBS1 protein, which are present in a large superfamily of proteins, and are recognized as major mediators of processes related to cell-cycle checkpoint and DNA repair. This review will focus on the current state-of-knowledge regarding the correlation between carriers of NBS1 gene mutations and the proneness to the development of malignant tumours.

Chromosome Instability and Nibrin Protein Variants in NBS Heterozygotes

The frequency of spontaneous chromosome abnormalities in peripheral blood lymphocytes and the X-ray G2 sensitivity in lymphoblastoid cell lines (LCL) have been evaluated in heterozygous subjects from three unrelated Nijmegen Breakage Syndrome (NBS) families, characterised by different mutations in the NBS1 gene. In all the 13 NBS heterozygotes analysed, we found spontaneous chromosome instability consisting in chromosome and chromatid breakages and rearrangements, while radiosensitivity was similar to that of control LCLs in seven out of eight tested NBS heterozygotes. The densitometric analysis of nibrin by immunoblotting indicated only a slight reduction in some of the LCLs from NBS carriers, whereas the immunoprecipitation assay appears a more reliable tool to detect NBS carriers. By means of immunoprecipitation, we investigated two homozygous and four heterozygous subjects. In the cells of the NBS patient 668, with the mutation 900del25, an alternative form of nibrin with a molecular weight of approximately 55 kDa has been detected. This variant protein, together with the normal p95, was also found in the LCL 34 established from a carrier of the same family. Signals of nibrin with a molecular weight lower than 95 kDa, but higher than that observed in LCLs 668 and 34, were detected also in three carriers from the family with mutation 835del4.

Peroxynitrite—An ugly biofactor?

Cellular damage occurring under oxidative conditions has been ascribed mainly to the formation of peroxynitrite (ONOOH/ONOO−) that originates from the reaction of NO• with O2•−. The detrimental effects of peroxynitrite are exacerbated by the reaction with CO2 that leads to ONOOC(O)O−, which further decays to the strong oxidant radicals NO2• and CO3•−. The reaction with CO2, however, may redirect peroxynitrite specificity. An excessive formation of peroxynitrite represents an important mechanism contributing to the DNA damage, the inactivation of metabolic enzymes, ionic pumps, and structural proteins, and the disruption of cell membranes. Because of its ability to oxidize biomolecules, peroxynitrite is implicated in an increasing list of diseases, including neurodegenerative and cardiovascular disorders, inflammation, pain, autoimmunity, cancer, and aging. However, peroxynitrite displays also protective activities: (i) at high concentrations, it shows anti‐viral, anti‐microbial, and anti‐parasitic actions; and (ii) at low concentrations, it stimulates protective mechanisms in the cardiovascular, nervous, and respiratory systems. The detrimental effects of peroxynitrite and related reactive species are impaired by (pseudo‐) enzymatic systems, mainly represented by heme‐proteins (e.g., hemoglobin and myoglobin). Here, we report biochemical aspects of peroxynitrite actions being at the root of its biomedical effects.

Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.

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.

In utero exposure to di-(2-ethylhexyl) phthalate affects liver morphology and metabolism in post-natal CD-1 mice

The plasticizer di-(2-ethylhexyl)phthalate (DEHP) affects reproductive development, glycogen and lipid metabolism. Whereas liver is a main DEHP target in adult rodents, the potential impact on metabolic programming is unknown. Effects of in utero DEHP exposure on liver development were investigated upon treatment of pregnant CD-1 mice on gestational days (GD)11-19. F1 mice were examined at post-natal days 21 (weaning) and 35 (start of puberty): parameters included liver histopathological, immunocytochemical and alpha-fetoprotein (AFP) gene expression analyses. In utero DEHP exposure altered post-natal liver development in weanling mice causing significant, dose-related (i) increased hepatosteatosis, (ii) decreased glycogen storage, (iii) increased beta-catenin intracytoplasmic localization (females only). At puberty, significantly decreased glycogen storage was still present in males. A treatment-induced phenotype was identified with lack of glycogen accumulation and intracytoplasmic localization of beta-catenin which was associated with increased AFP gene expression. Our findings suggested that DEHP alters post-natal liver development delaying the programming of glycogen metabolism.

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.

Hsp90: A New Player in DNA Repair?

Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis), transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR) pathways that include: (i) cell cycle arrest; (ii) transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii) triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.