Human genes containing polymorphic trinucleotide repeats.

Expansions of trinucleotide repeats within gene transcripts are responsible for fragile X syndrome, myotonic dystrophy and spinal and bulbar muscular atrophy. To identify other human genes with similar features as candidates for triplet repeat expansion mutations, we screened human cDNA libraries with repeat probes and searched databases for transcribed genes with repeats. From both strategies, 40 genes were identified and 14 characterized. Five were found to contain repeats which are highly polymorphic including the N-cadherin, BCR, glutathione-S-transferase and Na+/K+ ATPase (beta-subunit) genes. These data demonstrate the occurrence of other human loci which may undergo this novel mechanism of mutagenesis giving rise to genetic disease.

FMR1 protein: conserved RNP family domains and selective RNA binding.

Fragile X syndrome is the result of transcriptional suppression of the gene FMR1 as a result of a trinucleotide repeat expansion mutation. The normal function of the FMR1 protein (FMRP) and the mechanism by which its absence leads to mental retardation are unknown. Ribonucleoprotein particle (RNP) domains were identified within FMRP, and RNA was shown to bind in stoichiometric ratios, which suggests that there are two RNA binding sites per FMRP molecule. FMRP was able to bind to its own message with high affinity (dissociation constant = 5.7 nM) and interacted with approximately 4 percent of human fetal brain messages. The absence of the normal interaction of FMRP with a subset of RNA molecules might result in the pleiotropic phenotype associated with fragile X syndrome.

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.

A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.

The fragile X mental retardation protein inhibits translation via interacting with mRNA.

Fragile X syndrome is a frequent form of inherited mental retardation caused by functional loss of the fragile X mental retardation protein, FMRP. The function of FMRP is unknown, as is the mechanism by which its loss leads to cognitive deficits. Recent studies have determined that FMRP is a selective RNA-binding protein associated with polyribosomes, leading to the hypothesis that FMRP may be involved in translational regulation. Here we show that purified recombinant FMRP causes a dose-dependent translational inhibition of brain poly(A) RNA in rabbit reticulocyte lysate without accelerated mRNA degradation. In our translation reaction FMRP interacts with other messenger ribonucleoproteins and pre-exposure of FMRP to mRNA significantly increased the potency of FMRP as a translation inhibitor. Translation suppression by FMRP is reversed in a trans-acting manner by the 3'-untranslated portion of the Fmr1 message, which binds FMRP, suggesting that FMRP inhibits translation via interacting with mRNA. Consistently FMRP suppresses translation of the parathyroid hormone transcript, which binds FMRP, but not the beta-globin transcript, which does not bind FMRP. Moreover, removing the FMRP-binding site on a translation template abolishes the inhibitory effect of FMRP. Taken together, our results support the hypothesis that FMRP inhibits translation via interactions with the translation template.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Gentamicin diffusion across hydrogel bandage lenses and its kinetic distribution on the eye.

Soft contact lenses have been used as therapeutic bandages to aid epithelial healing following pentrating keratoplasty. Often the hydrogel lenses are used in conjunction with topical medications such as gentamicin. A reported complication is the persistence of infectious ulcers even though the eye is being treated with topical antibiotics. The purpose of this study was to measure the gentamicin diffusion coefficients for some hydrogel bandage lenses and to design a kinetic model to estimate the drug distribution on an eye covered with a hydrogel contact lens. The model includes the hydrogel diffusion coefficients and literature values for tear production, tear exchange per blink around the edge of a lens, fit, etc. From the computer generated data, it can be shown that the permeability of gentamicin sulfate through the Saulfon-80 hydrogel lens on a normal eye was only 0.002% of the amount of the drug under the contact lens after 10 minute intervals of topical drug application. The important drug distribution pathway was around the edge of the lens.

Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome.

The post-translational modification of proteins by covalent attachment of ubiquitin targets these proteins for degradation by the proteasome. An astounding number of proteins are involved in ubiquitination and deubiquitination of proteins. The pathways are combinatorial, and selectivity of proteolysis will depend strongly on the exact combination of ubiquitinating and deubiquitinating enzymes present at any time. In addition to temporal control, it is likely that these modifications are also regulated spatially. In this review, we discuss the regulation of ubiquitination by enzymes of this pathway and highlight some of the outstanding problems in understanding this regulation.

Roles of ubiquitinylation in proteolysis and cellular regulation.

Most eukaryotic organisms respond to starvation, nutrient deprivation, and/or stress by increasing the rates of intracellular proteolysis. The amino acids released may be reutilized for synthesis of important proteins, or directly for the production of energy. This enhanced proteolysis is also required for repair of cellular damage due to environmental insults such as heat shock, free radicals, viral infection, or mutation. Finally, intracellular proteolysis is important in determining the steady-state levels of a wide variety of regulatory proteins, particularly those regulating the cell cycle. The ubiquitin-dependent proteolytic system participates in all of these functions. In spite of its cytoplasmic localization, this system is selective and acts only on a limited set of substrates. This review discusses the mechanisms of this selectivity and the potential roles of ubiquitin-dependent proteolysis.

Protein ubiquitination: a regulatory post-translational modification.

The covalent attachment of ubiquitin to a variety of cellular proteins (ubiquitination) is a common post-translational modification in eukaryotic cells. Little is known about the function of these modifications in either the normal or the pathological state. The characteristics of ubiquitination in the nucleus, the cytoplasm, and on the plasma membrane are reviewed and discussed here. Also reported are studies on the enzymes which metabolize ubiquitin, using the ubiquitin-dependent proteolysis system as a model. Four enzymes which specifically recognize ubiquitin and hydrolyze carboxyl-terminal derivatives of ubiquitin have been partially purified from bovine thymus. These are thiol-containing proteases which will also release ubiquitin from ubiquitin-protein conjugates. The presence of these deconjugating enzymes and the proteases in the cytoplasm suggests that there is a partition of conjugates between proteolysis and deconjugation. To study the factors which determine the relative rates of proteolysis versus deconjugation, we have developed a general method of synthesizing large amounts of pure ubiquitin-protein conjugates. The structure/function relationships of ubiquitin have been probed by chemically modifying ubiquitin and examining its activity in the protein degradation system. These studies have identified regions of the ubiquitin molecule which are recognized by the enzymes of the proteolysis system, established that the molecule can be altered and used as a probe of such systems and will guide the design of site-directed mutant ubiquitins in order to more fully define the recognition sites on the ubiquitin molecule. It is likely that studies of these types will lead to an understanding of the molecular interactions required for proper ubiquitin function and allow design of drugs which could be useful in understanding the role of ubiquitination and its importance in normal and pathological states.

Regulation of ubiquitin-dependent processes by deubiquitinating enzymes.

An astounding number of important regulatory and structural proteins are subject to modification by the attachment of ubiquitin or ubiquitin-like proteins. This modification acts as a targeting signal, delivering the modified protein to different locations in the cell and modifying its activity, macromolecular interactions, or half-life. Deubiquitination, or the removal of this modification, is being recognized as an important regulatory strategy. This reaction is catalyzed by processing proteases known as deubiquitinating enzymes (DUBs). More than 60 DUBs are already known, although little is known about their biological roles. This review concentrates on recent findings and new insights into this fascinating class of enzymes.

Ubiquitin-dependent signaling: the role of ubiquitination in the response of cells to their environment.

The response of a cell to its external environment requires rapid and significant alteration of protein amount, localization and/or function. This regulation involves a complex combination of processes that control synthesis, localization and degradation. All of these processes must be properly regulated and are often interrelated. Intracellular proteolysis is largely accomplished by the ubiquitin-dependent system and has been shown to be required for growth control, cell cycle regulation, receptor function, development and the stress response. Substrates subject to regulated degradation by this system include cyclins and cyclin-dependent kinase inhibitors, tumor suppressors, transcription factors and cell surface receptors. In addition, proteins that are damaged by oxidation or that are improperly folded or localized are substrates whose degradation by this system often leads to antigen presentation on the surface of the cell in the context of Class I major histocompatibility complex molecules. A very large body of work in the last fifteen years has shown that degradation by this system requires the covalent attachment of a small protein called ubiquitin and that this modification serves to direct target proteins for degradation by a 26S proteolytic particle, the proteasome. Thus, the attachment of the ubiquitin domain is of vital importance in regulating normal growth and differentiation, as well as in defending against cellular damage caused by xenobiotics, environmental insults, infection and mutation. This review focuses on the role of ubiquitination in the cellular signaling pathways that deal with these external influences.

Immobilization of proteins via arginine residues.

A new method for activating polyacrylamide beads to bind proteins via arginine residues is described. The linking reagent, 4-(oxyacetyl)phenoxyacetic acid (OAPA), has been synthesized and characterized. OAPA reacts with arginine or N alpha-acetyl-L-arginine with a stoichiometry of 2 to 1. As expected for an arginine-specific reagent, OAPA inactivates horse liver alcohol dehydrogenase in a time-dependent manner, with the rate of this inactivation decreasing sixfold in the presence of 1 mM NADH. The presence of the carboxyl group in the linking reagent allows efficient coupling to aminated polyacrylamide beads. These derivatized beads are capable of binding various proteins via arginine residues in a time- and pH-dependent manner. Capacities range from less than 0.5 mg/ml to greater than 11 mg/ml, depending on the protein. The proteins are bound in a stable linkage, and preblocking the beads with either arginine or N alpha-acetyl-L-arginine eliminates all protein binding. Preblocking of the protein ubiquitin with OAPA reduces binding to a level compatible with the amount of underivatized ubiquitin remaining. The specificity, water solubility, negative charge, and linking ability of OAPA make it an especially valuable tool, both as a protein-modification reagent and as a linking reagent in preparing specialized affinity chromatographic media.

Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus.

In vivo, ubiquitin exists both free and conjugated through its carboxyl terminus to the alpha- and epsilon-amino groups of a wide variety of cellular proteins. Ubiquitin carboxyl-terminal hydrolytic activity is likely a necessary step in the regeneration of the ubiquitin cofactor from ubiquitin-protein conjugates. In addition, this type of activity is required to generate the active, monomeric ubiquitin from the only known gene products: the polyprotein precursor and various ubiquitin fusion proteins. Thus, this activity is of vital importance to systems that utilize ubiquitin as a cofactor. A generic substrate, ubiquitin ethyl ester, was previously developed [Wilkinson, K. D., Cox, M. J., Mayer, A. N., & Frey, T. (1986) Biochemistry 25, 6644-6649] and utilized here to monitor the fractionation of these activities from calf thymus. By use of a rapid HPLC assay, four distinct, ubiquitin-specific esterases were identified and separated. A previously undescribed activity has been resolved and characterized, in addition to the bovine homologue of ubiquitin carboxyl-terminal hydrolase purified from rabbit reticulocytes. Two other activities resemble deconjugating activities previously detected in crude extracts but not previously purified. These activities appear to form a family of mechanistically related hydrolases. All four activities are inhibited by iodoacetamide, indicating the presence of an essential thiol group, and are inhibited to various extents by manganese. All have specific ubiquitin binding sites as judged by the low observed Km values (0.6-30 microM). The carboxyl-terminal aldehyde of ubiquitin is a potent inhibitor of these enzyme activities, with Ki values approximately 1000-fold lower than the respective Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli.

Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.

Glucose exchange and catalysis by two crystalline hexokinase x glucose complexes. Evidence for an obligatory ATP-dependent conformational change in catalysis.

Hexokinase PI x glucose crystals grown with radiolabeled glucose under conditions similar to those used for x-ray diffraction studies (Bennett, W.S., Jr., and Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852) have been shown to contain 1 mol of tightly bound glucose. These crystals exchange all of this glucose in a single exponential process (kobs = 0.7 min-1). In the crystalline form they are, however, inactive and do not catalyze formation of any bound glucose-6-P, suggesting that lattice forces prevent catalysis. A new catalytically active E x glucose complex has been crystallized in the presence of glucose and ADP, a competitive inhibitor of ATP. These crystals readily lose ADP upon washing with concentrated (NH4)2SO4. They exchange all of the tightly bound glucose more slowly than the form grown in the absence of ADP (kobs = 0.05 min-1). Addition of MgATP to the suspension in ammonium sulfate results in rapid conversion of half of the bound glucose to bound glucose 6-phosphate followed by further reaction as products are released. This agrees with the previously measured equilibrium constant of unity for enzyme-bound phosphoryl transfer catalyzed in solution (Wilkinson, K.D., and Rose, I.A. (1979) J. Biol. Chem. 254, 12567-12572). These results indicate that the two E x glucose crystals are distinguished by a nucleotide-dependent conformational difference, which is stabilized by lattice forces. The active crystals allow the facile dissociation of the ADP used to induce the change. This conformational change appears to be pevented in the E x glucose crystals and to be necessary to produce the active ternary complex.

NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction.

A unique form of inhibition by NADH and partial reversal by NAD+ has been demonstrated with Escherichia coli lipoamide dehydrogenase. Substrate inhibition by NADH is consistent with its reduction of the active two-electron reduced enzyme intermediate to the inactive four-electron reduced form. NAD+ partially overcomes this inhibition by mass action reversal of this reduction. NAD+ activation is only partial since the presence of both NAD+ and NADH forces the accumulation of two binary enzyme-pyridine nucleotide complexes. These are intermediates in the two-electron to four-electron reduction of the enzyme and thus are not on the catalytic pathway. NAD+ is also shown to inhibit by binding to the oxidized enzyme to give a dead-end complex. From the steady state rate equations, it is apparent that the degree of inhibition will depend on the oxidation-reduction potential for two- to four-electron reduction of the enzyme. Thus, the wide variation in the severity of NADH inhibition between the E. coli and pig heart enzymes is explained by quantitative differences in the basic lipoamide dehydrogenase mechanism. A possible physiological role for this type of inhibition as a mechanism of control in E. coli is discussed.

Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.

It was previously shown that ubiquitin is very similar to the polypeptide cofactor of the ATP-dependent protein degradation system from rabbit reticulocytes (Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Biol. Chem. 255, 7529-7532). We have extended this work to show that the peptic peptide maps are identical for bovine ubiquitin and the polypeptide cofactor isolated from human erythrocytes. It was noted however that ubiquitin preparations were less active in stimulating proteolysis than preparations of the polypeptide cofactor. This decreased activity has been shown to be due to the presence of an inactive form of ubiquitin in some preparations. The two forms of ubiquitin are separable by high performance liquid chromatography. The active form of ubiquitin has the COOH-terminal sequence -Arg-Gly-Gly at residues number 74 to 76. The inactive form terminates in -Arg74 as previously reported in the sequence studies of ubiquitin. Limited tryptic digestion of active ubiquitin yields the inactive, later eluting form and the dipeptide glycylglycine. This preteolytic cleavage apparently occurs during purification from most tissues. We thus propose reserving the term ubiquitin for the intact 76-amino acid sequence and designating the 74-amino acid sequence as ubiquitin-t to indicate its derivation by a tryptic-like protease cleavage. This 76-residue sequence is consistent with the covalent structure of protein A-24, a conjugate where carboxyl group of the COOH-terminal glycylglycine of ubiquitin is linked by an amide bond to the epsilon-amino group of Lys-119 of histone H2A. Thus, the structural requirements of the protein and ubiquitin molecules are identical for formation of protein A-24 and for forming the covalent conjugates thought to be intermediates in ATP-dependent protein degradation.