Agnieszka Krakowiak

Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding

The FHIT gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the active-site histidine of which is not required for tumor suppression. To provide a rigorous test of the hypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding and/or hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in kcat and increases as large as 30-fold in KM. Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with KM. Mutants with 2- and 7-fold increases in KM significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in KM retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.

Coordinate Expression of NADPH-dependent Flavin Reductase, Fre-1, and Hint-related 7meGMP-directed Hydrolase, DCS-1

A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors. Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear. To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme. We used the novel substrate, 7meGpppBODIPY, to demonstrate that DCS-1 has low micromolar specificity for guanine ribonucleotides with the 7me modification, whereas trimethylated G substrates are poor competitors. Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases. These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts. We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have a nearly identical expression profile in human tissues. Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure. Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses.

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.

Stereochemical course of Escherichia coli RNase H

A new enzymatic method has allowed the assignment of the stereochemistry of E. coli RNase‐H‐assisted hydrolysis of RNA labelled within the scissile bond with (Rp)‐phosphorothioate. This method is based on a stereospecific, two‐step enzymatic conversion of cytidine 5′‐[18O]phosphorothioate into the corresponding 5′‐α‐[18O]thiotriphosphate, which is then further used for stereospecific transfer of cytidine 5′‐[18O]phosphorothioate to the 3′‐OH group of a short oligonucleotide with the aid of terminal deoxyribonucleotidyl transferase. Matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry of the resulting elongated primer revealed that RNase‐H‐assisted hydrolysis proceeds with inversion of configuration at the phosphorus atom. This result is discussed in the context of current knowledge of the architecture of the active site of the enzyme.

Helicobacter pylori Infection and Family History of Gastric Cancer Decrease Expression of FHIT Tumor Suppressor Gene in Gastric Mucosa of Dyspeptic Patients

Background:  The expression of a fragile histidine triad (FHIT) protein is lost in stomach tumors. The study aimed at determining whether FHIT expression is affected by Helicobacter pylori infection, strain virulence (vacA and cagA genes) and histopathological changes in the gastric mucosa of patients with functional dyspepsia having first‐degree relatives with gastric cancer.

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.

Expression of somatostatin receptor subtype 3 in the gastric mucosa of dyspeptic patients in relation to Helicobacter pylori infection and a family history of gastric cancer

Background and Aim:  The cytotoxic activity of Helicobacter pylori contributes significantly to the pathogenesis of gastric carcinoma. A preliminary study suggested that somatostatin receptor subtype 3 (SSTR3) might play a role in cell apoptosis and the growth of gastric cancer. The aim of the present study was to determine the influence of H. pylori infection and a family history of gastric cancer on the expression of SSTR3 in the gastric mucosa of non‐cancer patients with dyspepsia.

Searching for novel scaffold of triazole non-nucleoside inhibitors of HIV-1 reverse transcriptase

Abstract Azoles are a promising class of the new generation of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). From thousands of reported compounds, many possess the same basic structure of an aryl substituted azole ring linked by a thioglycolamide chain with another aromatic ring. In order to find novel extensions for this basic scaffold, we explored the 5-position substitution pattern of triazole NNRTIs using molecular docking followed by the synthesis of selected compounds. We found that heterocyclic substituents in the 5-position of the triazole ring are detrimental to the inhibitory activity of compounds with four-membered thioglycolamide linker and this substitution seems to be viable only for compounds with shorter two-membered linker. Promising compound, N-(4-carboxy-2-chlorophenyl)-2-((4-benzyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl)acetamide, with potent inhibitory activity and acceptable aqueous solubility has been identified in this study that could serve as lead scaffold for the development of novel water-soluble salts of triazole NNRTIs.

High-resolution X-ray structure of the rabbit histidine triad nucleotide-binding protein 1 (rHINT1)–adenosine complex at 1.10 Å resolution

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine-triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the first complete structure of the rabbit HINT1-adenosine complex is reported at 1.10 Å resolution, which is one of the highest resolutions obtained for a HINT1 structure. The final structure has an R(cryst) of 14.25% (R(free) = 16.77%) and the model exhibits good stereochemical qualities. A detailed analysis of the atomic resolution data allowed an update of the details of the protein structure in comparison to previously published data.

Crystallographic studies of the complex of human HINT1 protein with a non-hydrolyzable analog of Ap4A.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine triad proteins superfamily. HINT1 plays an important role in various biological processes, and it has been found in many species. Here, we report the first structure (at a 2.34Å resolution) of a complex of human HINT1 with a non-hydrolyzable analog of an Ap4A dinucleotide, containing bis-phosphorothioated glycerol mimicking a polyphosphate chain, obtained from a primitive monoclinic space group P21 crystal. In addition, the apo form of hHINT1 at the space group P21 refined to 1.92Å is reported for comparative studies.