The crosstalk between EGF,IGF, and Insulin cell signaling pathways - computational and experimental analysis

Background  

Cellular response to external stimuli requires propagation of corresponding signals through molecular signaling pathways. However, signaling pathways are not isolated information highways, but rather interact in a number of ways forming sophisticated signaling networks. Since defects in signaling pathways are associated with many serious diseases, understanding of the crosstalk between them is fundamental for designing molecularly targeted therapy. Unfortunately, we still lack technology that would allow high throughput detailed measurement of activity of individual signaling molecules and their interactions. This necessitates developing methods to prioritize selection of the molecules such that measuring their activity would be most informative for understanding the crosstalk. Furthermore, absence of the reaction coefficients necessary for detailed modeling of signal propagation raises the question whether simple parameter-free models could provide useful information about such pathways.     

Histidine triad nucleotide-binding protein 1 (HINT-1) phosphoramidase transforms nucleoside 5'-O-phosphorothioates to nucleoside 5'-O-phosphates

Nucleoside 5′-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5′-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5′-O-monophosphorothioate (AMPS) to 5′-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2′-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS ≥ CMPS > UMPS > dAMPS ≫ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released.     

Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation

Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys(525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys(525), is affected by the status of Cys(34). The disulfide forms of circulating albumin, albumin-Cys(34)-S-S-Cys and albumin-Cys(34)-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys(34)-SH. Although N-homocysteinylations of albumin-Cys(34)-SH and albumin-Cys(34)-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys(34) of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys(34)-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.     

A cyclo peptide activates signaling events and promotes growth and the production of the bone matrix

The interaction of bone cells and their underlying extracellular matrix impacts biological processes such as maintenance of tissue integrity. The biological recognition of the extracellular matrix by attached cells is mediated by the activity of integrins that recognize adhesive-specific domains. The most widely recognized adhesive motif is the RGD sequence, common to many of the adhesive matrix molecules. Here, we show that cyclo DFKRG which was previously selected to increase cell adhesion of human bone marrow stromal cells (HBMSC), increases both cell differentiation and mineralization through activation of tyrosine kinases, focal adhesion kinase (p(125)FAK) and Mitogen Activated Protein (MAP) kinases.     

Cardiomyocyte-specific endothelin A receptor knockout mice have normal cardiac function and an unaltered hypertrophic response to angiotensin II and isoproterenol

Even though endothelin is recognized as an important vasoregulatory molecule, the roles of endothelin receptors in specific cell types are not yet fully understood. Mice with a null mutation in endothelin A receptor gene (ET(A)) or in the gene of its ligand (endothelin 1) die neonatally due to craniofacial and cardiac abnormalities. This early lethality has in the past hindered studies on the role of endothelin in cardiovascular physiology and pathophysiology. To overcome this obstacle, we utilized the cre/loxP technology to generate mice in which the ET(A) gene could be deleted specifically in cardiomyocytes. The cre recombinase transgene driven by the alpha-myosin heavy-chain promoter deleted the floxed ET(A) allele specifically in the hearts of these mice, resulting in a 78% reduction in cardiac ET(A) mRNA level compared to wild-type controls. Cardiomyocyte-specific ET(A) knockout animals are viable and exhibit normal growth, cardiac anatomy, and cardiac contractility, as assessed by echocardiography. In addition, these animals exhibit hypertrophic and contractile responses to 10-day infusion of angiotensin II or isoproterenol similar to those observed in control animals. These results indicate that in adult mice cardiac ET(A) receptors are not necessary for either baseline cardiac function or stress-induced response to angiotensin II or isoproterenol.     

Urinary excretion of DNA repair products correlates with metabolic rates as well as with maximum life spans of different mammalian species

Using recently developed methodology, which includes HPLC prepurification followed by GC/MS with isotope dilution, we analyzed urinary excretion of possible repair products of oxidative DNA damage-8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and 5-(hydroxymethyl)uracil (5-HMUra)-in mammalian species that substantially differ in metabolic rate and longevity, namely, mice, rats, rabbits, dogs, pigs, and humans. We found highly significant, positive correlations between specific metabolic rates of the animals studied and their excretion rates for all the modifications analyzed with respective r values for the lesions of (8-oxoGua) r = .891, p < .01; (8-oxodG) r = .998, p < .001; and (5-HMUra) r = .949, p < .005. However, only 8-oxoGua significantly correlates negatively with maximum life span (MLSP) (r = -.928, p < .01). Despite substantial differences in MLSP between humans and pigs (120 and 27 years, respectively), the rates of excretion of all measured modifications were very similar. The urinary levels of all measured modifications found in our study for mouse and humans account respectively for about 34,000 and 2800 repaired events per average cell, per 24 h. It is therefore possible that the high metabolic rate in mice (or other short-lived animals) may be responsible for severe everyday oxidative DNA insults that may be accumulated faster than in long-lived species.