Angela K. Robinson

THE TUMOR SUPPRESSOR PROTEIN FHIT : A NOVEL INTERACTION WITH TUBULIN

FHIT (fragilehistidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5′,5′′′-P1,P3-triphosphate (Ap3A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap3A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K d values of 1.4 and 2.1 μm, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap3A hydrolase activity of Fhit.

Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.

Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography.

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.

The Growth-Suppressive Function of the Polycomb Group Protein Polyhomeotic Is Mediated by Polymerization of Its Sterile Alpha Motif (SAM) Domain

Background: The mechanism by which Sterile Alpha Motifs (SAMs) self-associate and polymerize to control protein function is unknown. Results: SAM polymerization in Polyhomeotic, a Polycomb group protein, is controlled by an unstructured linker sequence in Polyhomeotic. Conclusion: Polyhomeotic growth suppressive function is enhanced by increasing SAM polymerization. Significance: Functions of other SAM domain-containing proteins could be manipulated through their unstructured linkers. Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.

High-performance liquid chromatographic application of the hummel and dreyer method for the determination of colchicine—tubulin binding parameters

An application of the Hummel and Dreyer gel chromatography procedure modified for high-performance liquid chromatography has been used to determine the dissociation constant for the colchicine-tubulin interaction at 25 degrees C. The results obtained are compared with results of other equilibrium and non-equilibrium techniques and demonstrate that the initial interaction of colchicine with tubulin must be rapid and probably reversible. This rapid and sensitive technique, which does not require radioisotopes for measurement of the binding parameters, will be extremely useful for characterization of tubulin-ligand interactions.

Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe

An enzyme that catalyzes the asymmetric hydrolysis of Ap4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap4A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 microM for Ap4A. Both forms of the enzyme asymmetrically hydrolyze Ap4A to AMP and ATP as determined by HPLC. Ap4A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 microM. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap4A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50 microM to 20 mM, has no significant effect on Ap4A hydrolase activity.

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.

Three diadenosine 5′,5‴-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: A specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase

Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported.

Binding of colchicine to renal tubulin at 5°C

Previous reports in the literature state that the binding of colchicine to soluble tubulin is negligible at 0 – 4°C as measured by non-equilibrium binding methods. In contrast, we have detected significant binding of colchicine to tubulin at 5°C. Furthermore, for the first time equilibrium dialysis has been used to measure colchicine binding. The value of the dissociation constant was 1.8 μM at 5°C, and the stoichiometry of colchicine binding at 5°C equaled that at 37°C. Dissociation at 5°C of bound colchicine was negligible over a period of 23 h, and the estimated minimal half-time of dissociation was 150 h.

Identification of nucleic acid binding residues in the FCS domain of the polycomb group protein polyhomeotic.

Polycomb group (PcG) proteins maintain the silent state of developmentally important genes. Recent evidence indicates that noncoding RNAs also play an important role in targeting PcG proteins to chromatin and PcG-mediated chromatin organization, although the molecular basis for how PcG and RNA function in concert remains unclear. The Phe-Cys-Ser (FCS) domain, named for three consecutive residues conserved in this domain, is a 30-40-residue Zn(2+) binding motif found in a number of PcG proteins. The FCS domain has been shown to bind RNA in a non-sequence specific manner, but how it does so is not known. Here, we present the three-dimensional structure of the FCS domain from human Polyhomeotic homologue 1 (HPH1, also known as PHC1) determined using multidimensional nuclear magnetic resonance methods. Chemical shift perturbations upon addition of RNA and DNA resulted in the identification of Lys 816 as a potentially important residue required for nucleic acid binding. The role played by this residue in Polyhomeotic function was demonstrated in a transcription assay conducted in Drosophila S2 cells. Mutation of the Arg residue to Ala in the Drosophila Polyhomeotic (Ph) protein, which is equivalent to Lys 816 in HPH1, was unable to repress transcription of a reporter gene to the level of wild-type Ph. These results suggest that direct interaction between the Ph FCS domain and nucleic acids is required for Ph-mediated repression.