Genetic, biochemical, and crystallographic characterization of Fhit–substrate complexes as the active signaling form of Fhit

Pace, Helen C.; Garrison, Preston N.; Robinson, Angela K.; Barnes, Larry D.; Draganescu, Alexandra; Rösler, Ariel; Blackburn, G.M.; Siprashvili, Zurab; Croce, Carlo M.; Huebner, Kay; Brenner, Charles

doi:10.1073/pnas.95.10.5484

Cited by 137 publications

(197 citation statements)

References 21 publications

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“…According to that proposed mechanism, His98, the final His of the HIT motif, which aligns with Gln168 of GalT, makes a hydrogen bond with an α phosphate oxygen of an ApppA substrate. In our structures of wild-type and mutant Fhit bound to ApppA analog, IB2, which has a phosphorothio substitution on one α phosphate and substitution of a methylene group for the other α-β bridging oxygen (104), we observed His98 to be positioned to interact with the α-β bridging oxygen (72) and not an α phosphate oxygen like Gln168 of GalT (3). The distance between the His98 εN and the α-β bridging oxygen in the co-crystal structure of Fhit H96N with nonhydrolyzable ApppA was 2.5 Å (72), which is in the range of low barrier hydrogen bonds that are frequently found at enzyme active sites (105).…”

Section: Mechanistic Insights In the Hit Superfamily: Hydrolases Versmentioning

confidence: 89%

“…Second, Ap n A may signal through Fhit in an antiproliferative or pro-apoptotic manner that is lost with loss of Fhit. Third, the tumor suppressing function may be Ap n A-independent (72). Because the H96N mutant of Fhit had been shown to be extremely deficient in enzymatic activity (40) and yet functional in tumor suppression (59), it was important to characterize the nature of the biochemical defect.…”

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

“…This was clearly inconsistent with the idea that Fhit must reduce the concentration of Ap n A to function in tumor suppression. Because the H96N mutant retained a low micromolar K m for ApppA and was found crystallographically to bind nonhydrolyzable ApppA in a manner nearly identical to that of wild-type Fhit, it was argued that the tumor suppressing function of Fhit may depend on forming an Ap n A complex (72). Furthermore, the crystallographically defined Fhit dimer surface that becomes filled with two Ap n A molecules was proposed as a possible site of effector interactions (72).…”

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

“…Because the H96N mutant retained a low micromolar K m for ApppA and was found crystallographically to bind nonhydrolyzable ApppA in a manner nearly identical to that of wild-type Fhit, it was argued that the tumor suppressing function of Fhit may depend on forming an Ap n A complex (72). Furthermore, the crystallographically defined Fhit dimer surface that becomes filled with two Ap n A molecules was proposed as a possible site of effector interactions (72). The critical test of this model depends either on discovery of an effector that binds Fhit-Ap n A, potentially in a manner that retards hydrolysis, or showing that Fhit mutants that are defective in substrate-binding are poor tumor suppressors (72).…”

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

“…Furthermore, the crystallographically defined Fhit dimer surface that becomes filled with two Ap n A molecules was proposed as a possible site of effector interactions (72). The critical test of this model depends either on discovery of an effector that binds Fhit-Ap n A, potentially in a manner that retards hydrolysis, or showing that Fhit mutants that are defective in substrate-binding are poor tumor suppressors (72). These are both active research areas.…”

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

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Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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HIT (histidine triad)1 proteins, named for a motif related to the sequence HφHφHφφ, (φ a hydrophobic amino acid) are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylylsulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding-site. The potential roles of ASW and Hint in avian sexual development are discussed in an accompanying manuscript. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases. Mammalian Hint and GalT Structurally Define the HIT SuperfamilyGalactose-1-phosphate uridylyltransferase (GalT), the second enzyme in the Leloir pathway of galactose utilization (1), was crystallized and its structure determined to understand the mechanistic basis for transferring UMP between glucose-1-phosphate and galactose-1-phosphate (2-4). Histidine triad nucleotide-binding protein (Hint), purified as a purine nucleoside/nucleotide-binding protein from rabbit heart cytosol (5) and shown to have a widely conserved sequence (6), was crystallized and its structure determined to establish whether the conserved sequence formed the basis for a new mode of nucleotide-binding (7). GalT (2) and Hint are both dimers (8) but the GalT monomer is more than twice the size of the Hint dimer. Though GalT (9) and Hint (6) homologs could be cloned by similarity and the crystal structures (2,8) co-existed in protein databases, mutual similarity was not noted until inspection of the rabbit Hint crystal structure (7) and computational analysis (10) indicated that a GalT monomer and a Hint dimer can be superimposed and that their nucleotide-binding sites retain some of the same residues for binding a nucleoside monophosphate (7) (Figure 1). The level of sequence similarity of Hint and GalT is difficult to distinguish from n...

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Section: Mechanistic Insights In the Hit Superfamily: Hydrolases Versmentioning

confidence: 89%

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

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Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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The histidine triad superfamily of nucleotide-binding proteins

et al. 1999

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Histidine triad (HIT) proteins were until recently a superfamily of proteins that shared only sequence motifs. Crystal structures of nucleotide-bound forms of histidine triad nucleotide-binding protein (Hint) demonstrated that the conserved residues in HIT proteins are responsible for their distinctive, dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites. Hint-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Hint homologs are intracellular receptors for purine mononucleotides whose cellular function remains elusive. Fhit homologs bind and cleave diadenosine polyphosphates (Ap(n)A) such as ApppA and AppppA. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues. In invertebrates, Fhit homologs are encoded as fusion proteins with proteins related to plant and bacterial nitrilases that are candidate signaling partners in tumor suppression.

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Tripodale, „supergeladene” Analoga von Adenosinnucleotiden: Inhibitoren des Fhit-Proteins, einer Ap3A-Hydrolase

et al. 1999

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Methantrisphosphonsäuren bieten eine Verzweigungsstelle für synthetische Nucleotidanaloga, die entweder zu neuartigen tripodalen Nucleotiden führen oder zum Einbau zusätzlicher negativer Ladung in lineare Analoga eines Stammnucleotids genutzt werden können (im Bild ist dies für ATP und Diadenosintetraphosphat (Ap4A) gezeigt). Diese Verbindungen sollten von großem Nutzen sein, da sie als kompetitive Inhibitoren deutlich zwischen dem Tumorsuppressorprotein Fhit und einer anderen ApnA‐Pyrophosphohydrolase unterscheiden. X = H, Cl, F.

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Genetic, biochemical, and crystallographic characterization of Fhit–substrate complexes as the active signaling form of Fhit

Abstract: ABSTRACT

Cited by 137 publications

References 21 publications

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

The histidine triad superfamily of nucleotide-binding proteins

Tripodale, „supergeladene” Analoga von Adenosinnucleotiden: Inhibitoren des Fhit-Proteins, einer Ap3A-Hydrolase

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