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J. Biol. Chem., Vol. 280, Issue 47, 38889-38897, November 25, 2005
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Reflections

From Polynucleotide Phosphorylase to Neurobiology

Uriel Z. Littauer

From the Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel

In the fall of 1944, I enrolled at the Hebrew University of Jerusalem. The student body numbered about 700 and the choice of faculties was somewhat limited. My hope was to study medicine, but the plans to open a medical school were still at the drawing board stage. I therefore chose to study chemistry with biochemistry and bacteriology as minor subjects, which I thought I would need later if I went into medicine. As employment opportunities were limited in British Mandatory Palestine, it was hoped that university training would help me get a job in the food technology industry or at the Dead Sea potash industry.

When I started as a student, the structure and function of DNA and RNA were not known. Thymonucleic acid (DNA) had been isolated from thymus and pus, and zymonucleic acid (RNA) was found in yeast. The tetranucleotide hypothesis for DNA suggested by Phoebus Aaron Theodor Levene still prevailed, and there were speculations about branched chain as distinct from linear structures for RNA. Nucleic acids were also considered as nothing more than a storehouse of phosphorus for the varying requirements of the cell. However, the role of DNA in bacterial transformation, demonstrated by Oswald Theodore Avery, Colin MacLeod, and Maclyn McCarty in 1944, was discussed extensively during our bacteriology courses. Within a few years, I had the good fortune to enter the nucleic acid field where progress had evolved dramatically.

In June 1949, upon the completion of my studies at the Hebrew University of Jerusalem, Professor Ernst David Bergmann, the Scientific Director of the Weizmann Institute of Science, invited me to join him as his Ph.D. student. I gladly accepted and moved to Rehovot, which was then a small village with about 7000 inhabitants. Ernst Bergmann was an organic chemist, a former student of the noted German chemist, Wilhelm Schlenk, and co-worker of Professor Chaim Weizmann, the first President of the State of Israel. Bergmann had a dynamic and brilliant personality with an encyclopedic knowledge of chemistry and a broad interest in science (1). For my doctoral dissertation, Bergmann recommended I choose a subject close to Chaim Weizmann's scientific interests, namely the mechanism of pentose fermentation in bacteria. While I was in the advanced stages of my doctoral work, Sol Spiegelman from the Department of Microbiology of the University of Illinois, Urbana came to Rehovot to consider an offer that had been made to him to join the Weizmann Institute. Spiegelman suggested that for my postdoctoral studies I contact Arthur Kornberg, Head of the Department of Microbiology of the Washington University School of Medicine in St. Louis, Missouri. Kornberg had just moved from the National Institutes of Health (NIH) and was recruiting people for his new department. I was greatly impressed by Arthur Kornberg's early publications on coenzyme and nucleotide synthesis and decided to write and ask if he would accept me as a postdoctoral fellow. Spiegelman offered to talk to Kornberg on my behalf, and on the strength of his recommendation, I had the good fortune to be accepted and to receive a fellowship from the Dazian Foundation.

In March 1955, I arrived in St. Louis to join Arthur Kornberg's laboratory. He proposed that I try to construct a cell-free system that would catalyze the synthesis of RNA. As substrate, I used 14C-labeled ATP, which I had to synthesize myself from 14C-labeled adenine by a series of enzymatic reactions because commercially labeled nucleotides were not available then. Within a short time, I was able to construct a cell-free system from Escherichia coli cells that converted 14C-labeled ATP to an acid-insoluble polyribonucleotide. Moreover, the addition of adenylate kinase (myokinase) to the system increased the rate of the reaction. Although the activity was barely detectable, Kornberg thought that I should attempt to characterize the polynucleotide-synthesizing system. While making rapid progress in purifying the E. coli enzyme, we learned from Herman Kalckar, an eminent Danish biochemist who came for a visit, that Marianne Grunberg-Manago and Severo Ochoa at New York University had independently discovered an activity, similar to ours, in extracts of Azotobacter vinelandii (A. agilis). The enzyme was named polynucleotide phosphorylase (PNPase) and was shown to convert nucleoside diphosphates into polynucleotides. Although we were disappointed by the news of their findings, we decided to continue our studies with the purified E. coli enzyme. Acting on this new information, we shifted to using ADP rather than ATP and found it to be the preferred substrate in our system (2, 3).

PNPase was the first enzyme to be discovered that catalyzes the de novo synthesis of polyribonucleotides with a 3',5'-phosphodiester bond, and its discovery stimulated a considerable number of investigations. The cumulative studies have established that in the forward reaction long polyribonucleotides (pN)n are synthesized in a processive fashion from various ribonucleoside diphosphates (ppN) with concomitant release of inorganic phosphate (Pi). Each of the four common ribonucleoside diphosphates can serve separately as a substrate for the polymerization reaction, leading to formation of homopolymers. Polymerization of a mixture of ribonucleoside diphosphates that contains different bases results in a random copolymer. The enzyme does not require a template and cannot copy one. In the reverse reaction, PNPase is a processive 3' to 5' exoribonuclease that catalyzes the stepwise phosphorolysis of single-stranded polyribonucleotides, liberating ribonucleoside diphosphates.

In the processive phosphorolysis of long chain polyribonucleotides, the enzyme tends to degrade a single chain to completion, releasing ribonucleoside diphosphates plus a resistant short oligoribonucleotide before commencing phosphorolysis of another chain. In contrast, short oligoribonucleotides are degraded by a random nonprocessive mechanism in which the enzyme dissociates from the substrate after the phosphorolysis of each nucleotide (reviewed in Refs. 4 and 5). PNPase was also found to catalyze an exchange reaction between free in organic phosphate and the {beta}-phosphate of several ribonucleoside diphosphates. This reaction is apparently a result of combined polymerization and phosphorolytic reactions that occur under equilibrium conditions (2-4, 6). Under suitable conditions, the enzyme also catalyzes the elongation of a primer oligoribonucleotide with a free 3'-terminal hydroxyl group (7, 8). At that early stage, we pointed out that it is not apparent how an enzyme that appears to polymerize the available ribonucleoside diphosphates in a random fashion produces the specific nucleotide composition of the RNA of a given species (3). Subsequent research in other laboratories showed that the cellular function of the enzyme is to degrade RNA and that RNA synthesis is catalyzed by DNA-directed RNA polymerase.

In the spring of 1956, the enzyme was already purified about 300-fold when Arthur informed me that Leon Heppel, then Head of the Section for Metabolic and Arthritis Diseases at the NIH, was interested in the enzyme. Leon then came for a visit of a few days to get acquainted with our purification procedure and assay systems. Leon, devoted to his scientific work, did not waste a minute and spent long hours into the night working in our laboratory. Prior to his return to Bethesda, I gave him some of our purified PNPase and an "activator" fraction. Toward the end of my stay in St.Louis, Alex Rich and Leon Heppel invited me to the NIH to acquaint them with my PNPase purification procedure and the synthesis of long polynucleotides. While there I also examined the phosphorolysis of several RNA preparations, and together with Gary Felsenfeld and Alex Rich we analyzed the various synthetic polyribonucleotides in the ultracentrifuge. The sedimentation data indicated molecular weights of about 400,000. They later obtained some excellent x-ray diffraction pictures from the polynucleotides. Heppel was very generous in providing me with samples of his RNA and enzyme collection, which I intended to use upon my return to the Weizmann Institute. In our experiments PNPase was found to readily catalyze the phosphorolysis of synthetic polyribonucleotides as well as high molecular weight RNA preparations, whereas commercial and low molecular weight RNA samples were not attacked to a significant extent (3). I suspected that the RNA samples were degraded products that were resistant to phosphorolysis by the enzyme.

Later, in Rehovot, these observations led me to develop a method for isolating intact bacterial and mammalian RNA. Analysis in the ultracentrifuge of the E. coli preparations revealed the existence of several types of RNA with sedimentation constants of 4.1, 16.5, and 23.7S. The two high molecular weight RNA components were separated from the low molecular weight fraction by ammonium sulfate precipitation and turned out to be derived from ribosomes. These were not trivial findings because a number of investigators considered ribosomal RNA (rRNA) as an aggregate of short polynucleotides and did not realize the inherent instability of RNA compared with DNA. As expected the high molecular weight rRNA components were efficiently phosphorolyzed by PNPase. On the other hand, transfer RNA (tRNA) preparations were attacked more slowly and to a limited extent (20-30%). Urea was found to increase the degree of breakdown of tRNA, indicating that the secondary structure of tRNA hinders the enzymatic attack (9, 10).

Having at hand intact rRNA preparations, I suggested to my good friend and colleague, Heini Eisenberg, that we collaborate in an attempt to determine their physical properties. At that time the structure of RNA, unlike that of double-stranded DNA, was unknown mainly because of the lack of undegraded RNA preparations and because the existence of several types of RNA was not yet realized. Early attempts to secure clear diffraction patterns of RNA had failed (11). We soon discovered the single-stranded nature of rRNA and the ways in which it differs from double-stranded DNA. Thus, viscosity and birefringence of flow measurements showed that rRNA is quite sensitive to increasing the ionic strength of the solution in contrast with double-stranded DNA, which shows a much smaller dependence on ionic strength concentrations. We proposed, therefore, that rRNA behaves as a flexible, contractile single-stranded coil and that each ribosomal subunit contains a single continuous uninterrupted RNA chain (reviewed in Ref. 10). Further experiments on rRNA were performed in collaboration with Robert Cox (a visiting scientist now at the National Institute for Medical Research, Mill Hill). Our studies showed a close correlation of the ionic strength dependence of optical rotation, optical density, and hydrodynamic properties. These early results indicated that rRNA possessed a significant secondary structure, a rather novel observation for its time. Several years later we demonstrated together with my graduate student, Inder M. Verma (now a Professor and leading molecular biologist at the Salk Institute), and Marvin Edelman (then a visiting scientist from Harvard Medical School) the presence of rRNA in mitochondria from several fungal species. We also showed that the mitochondrial rRNA possesses a unique ordered structure that differs from that of the homologous cytoplasmic rRNA (reviewed in Refs. 10 and 12). By this time, we had become interested in understanding the mechanisms that govern the regulation of tRNA and mRNA activity. We devised methods for the purification of specific tRNA species and studied the post-transcriptional modification of tRNA chains. In particular we were intrigued by the high content of modified bases in tRNA. We showed that methylated bases in tRNA are not likely to be essential for cell viability but depending on the type of base modification and position along the tRNA chain they may play a role in the fine tuning of tRNA activity (12).

The late Violet Daniel, my first graduate student (who later became a Weizmann Institute Professor of Biochemistry), joined my laboratory in 1957. She purified and characterized tRNA nucleotidyltransferase from rat liver. The enzyme was found to have an important role in the proofreading and repair of the universal 3'-CCA end of tRNA. Further experiments by the late Jacov Tal (until recently Professor and Head of the Virology Department at Ben-Gurion University) showed that the enzyme adds CMP to tRNA... N by a nonprocessive mechanism. Moreover, together with Violet and colleagues we developed a novel method that allowed monitoring the hybridization of individually labeled aminoacylated tRNA species with DNA. Using that method, we were able to reveal the presence of several unique T4 phage-coded tRNA species (13). Our discovery was well received during an EMBO workshop on tRNA that was organized by Sydney Brenner in Cambridge, UK, in March 1969. In another project that involved Violet Daniel, Jacques S. Beckmann, Sara Sarid, Jacob I. Grimberg, and Max Herzberg we were the first to isolate a tRNA gene (14).

Simultaneously, our studies with PNPase have continued. Together with Yosef Kimhi, another graduate student (who became later Vice President of Scientific Affairs, Yeda Co.), we purified further the E. coli enzyme. Evidence was obtained to support our hypothesis that the same enzyme catalyzes the nucleotide polymerization and the ADP-Pi exchange reaction (6). In addition, the intracellular distribution of PNPase was examined in E. coli cells. We showed that the major part of the enzyme activity (80%) is present in the soluble fraction; 10% of the total activity is bound to the cell membrane; and about 10% remains bound to washed ribosomes (15). Several years later Hermona (Mona) Soreq, a graduate student (now Professor and Head of the Institute of Life Sciences at the Hebrew University of Jerusalem), succeeded in purifying E. coli PNPase to homogeneity (16). The purified enzyme was virtually free of contaminating nucleases, which allowed us to use its 3'-exonucleolytic activity to determine the size and composition of the 3'-terminal sequences of RNA molecules and their function. Thus, with a molar excess of PNPase over the substrate a synchronous mode of phosphorolysis is established in which NDP molecules are sequentially released from the 3' terminus of the RNA chains (4, 17). Moreover, we observed that at 0 °C, the poly(A) tails of mRNA molecules are readily phosphorolyzed, whereas the deadenylated mRNA chains remain intact. Together with Uri Nudel, Raphael Salomon, and Michel Revel we found that globin poly(A)-free mRNA could still be translated in a Krebs ascites tumor cell-free extract (the nuclease-treated reticulocyte lysate cell-free system had not yet been developed). We also observed that at long periods of incubation, the rate of globin synthesis appeared to level off more sharply with deadenylated mRNA than with native mRNA (17). The in vitro systems survive no longer than 2 h and are inadequate for detection of long term effects of the poly(A) tail on mRNA stability. We, therefore, turned our attention to the use of Xenopus laevis oocytes and were fortunate to collaborate with Georges Huez and Gérard Marbaix from the Free University of Brussels, who were experts in the use of this relatively new system. To examine the functional stability (i.e. ability to be translated) of the mRNA, deadenylated mRNA samples were microinjected into the oocytes. The results showed that the rate of globin synthesis with poly(A)-free mRNA is considerably lower than with native mRNA, and this difference became more pronounced at longer periods of incubation (18). In subsequent experiments we were able to show that readenylation of poly(A)-free globin mRNA restores its functional stability. In retrospect, we were more than fortunate in choosing globin mRNA for our studies, as there are examples where the removal of poly(A) tracts from some other mRNA species does not affect their stability (reviewed in Ref. 19).

Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by Uri Nudel and Hermona Soreq. This was accomplished by partial phosphorolysis of mRNA with the purified E. coli PNPase. By varying the salt concentration and the time of incubation of the phosphorolysis reaction mixture, as well as performing oligo(dT)-cellulose chromatography at different temperatures, globin mRNA preparations with poly(A) tails of varying size were obtained. In collaboration with our Belgian colleagues, Gérard Marbaix, Georges Huez, Madeleine Leclercq, Evelyne Hubert, and Hubert Chantrenne, the functional stability of these molecules was examined in Xenopus oocytes. Globin mRNA molecules with a segment of 32 or more adenylate residues had equivalent functional stability, whereas those with less than 32 adenylate residues were 10-fold less stable. We suggested that a minimal size of the poly(A) segment is essential for attaching to poly(A)-binding proteins (PABP), thereby protecting the mRNA from nucleolytic degradation (20). This suggestion correlates well with the observation of Bradford Baer and Roger Kornberg that the minimal length of the poly(A) tail necessary for PABP binding is 27 residues (21). To account for the great variability among mRNA species, it was proposed that the 3'-untranslated region (3'-UTR) can modulate the affinity of PABP for the poly(A) segment, thus permitting control of the poly(A) stability in individual mRNA species (22). It is also apparent that the interaction between the poly(A) tail-PABP complex and cap-associated translation initiation factors may be important in maintaining the physical integrity of mRNA (23). Thus, there is a multitude of systems that use the poly(A) tract to control the expressions of specific mRNA species. The number of cis-acting elements and trans-acting factors regulating turnover of mRNA is increasing rapidly, and the complexity of these processes grows in parallel.

We also used the 3'-exonucleolytic activity of PNPase to determine the size of the poly(A) tails from various mRNA species (17, 20, 24). In other studies, Raphael Salomon and colleagues were able to examine the regulatory function of the 3'-region of tobacco mosaic virus (TMV) RNA. This was accomplished by subjecting TMV RNA molecules to limited phosphorolysis by PNPase (25). Additional applications of the 3'-exonucleolytic activity of the enzyme were developed for sequence analysis of short oligoribonucleotides (26). Finally, Gabriel Kaufmann (now Professor and Head of the Department of Biochemistry at Tel Aviv University) determined the substrate specificity of PNPase. The enzyme was found to direct the reversible addition of a single deoxynucleotidyl residue to ribooligonucleotide primers, whereas further addition of deoxynucleotidyl residues to the resulting product continued at a very slow rate (27). The enzyme was also found to phosphorolyze aminoacyl-tRNAs, thereby yielding aminoacyl-ADP and nucleoside diphosphates (28). These observations prompted us to investigate the properties of ribonucleoside diphosphate analogs modified in their sugar moiety as substrates for the enzyme. We suggested that blocking of NDPs at their 3'-hydroxyl function would yield "monofunctional" substrates to which only one residue may be added to an oligonucleotide primer, thus serving as chain terminators. The blocking group can be subsequently removed chemically from the oligonucleotide products, permitting a succession of single addition reactions. This procedure was employed for the stepwise synthesis of polyribonucleotides of defined sequence (26, 29). Combinations of these reactions and using T4 RNA ligase to ligate the synthesized oligonucleotides allowed the synthesis of appreciable long oligonucleotides. Kaufmann further adapted the use of this ligase for unique sequence insertions and alterations in tRNA anticodon loops (30).

PNPase has been the subject of numerous studies. It was employed as a tool for producing model nucleic acids and solving many important biological problems. Thus, establishing the genetic code was facilitated by the ability of PNPase to synthesize heteropolymers and triplet nucleotides. The advances made in the understanding of the physicochemical properties of polyribonucleotide chains and their hybridization reactions, as well as the synthesis of polynucleotide inducers of interferon, are further examples of the role played by the enzyme. I was glad to have had the opportunity to write a review, together with Marianne Grunberg-Manago, summarizing the voluminous studies on this enzyme that have accumulated over the years (5). More recent studies in several laboratories have revealed that the PNPase gene sequence is evolutionary conserved. It is widely distributed among a variety of aerobic, anaerobic, halophilic, and thermophilic bacteria. However, it is absent in all Archaea-sequenced genomes examined to date. It is also missing in some single-celled eukaryotes such as yeast but is present in animal eukaryotes. Thus, functional human PNPase was identified in an overlapping pathway screen to discover nuclear genes that displayed coordinated expression as a consequence of terminal differentiation and cellular senescence of human melanoma cells (31-33). The human PNPase is localized in the mitochondria (34) and in comparison with bacterial PNPase contains an extended N-terminal sequence that might serve as a mitochondrial import signal (35). Plants were shown to contain two PNPase species (36, 37). Although genes in the nucleus encode both PNPase species, their presence in plants is very likely the result of two separate horizontal transfer events. One PNPase functions in the chloroplasts while the second is found in the mitochondria.

The E. coli enzyme is a homotrimer (16, 38) and all three enzyme subunits participate in the phosphorolysis of each RNA chain. Thus, only one polyribonucleotide can be processed per trimer (16). In addition, Portier and colleagues showed that the E. coli PNPase subunit contains 711 amino acids and is coexpressed with the gene for ribosomal protein S15 as part of the rpsO-pnp operon. Two promoters have been identified in the operon: P1, situated upstream of rpsO, and P2, located in the intergenic region between rpsO and pnp (39, 40). It was also demonstrated that in E. coli cells, PNPase autoregulates its own synthesis post-transcriptionally in an RNase III-dependent manner (41). In addition, it was found that the steady-state levels of the enzyme increase at relatively low temperatures and are linked to the efficiency of the autocontrol mechanism (40). The deduced amino acid sequences of PNPase from different bacteria, as well as that from the nuclear genomes of plants and mammals, have been compared. They display similar structures composed of five distinguishable domains, each of them highly conserved. The protein consists of two core domains having different degrees of identity to E. coli RNase PH and an {alpha}-helical domain located between the two core domains. In addition, there are two adjacent C-terminal RNA-binding domains, KH and S1 (35, 42-44). Like PNPase, RNase PH is a 3' to 5' exonuclease that catalyzes phosphate-dependent degradation of RNA. The degradation targets of these enzymes in vivo, however, are different. While PNPase is mainly involved in the degradation of mRNA decay intermediates (45, 46) and is stalled by RNA tertiary structure (5), the primary function of RNase PH is thought to be the processing of 3'-ends of precursor tRNA molecules (47, 48). The trimeric structure of PNPase is also suggested from electron microscopic studies in which the enzyme appears as a triangular complex with a visible central hole (49). Moreover, the crystal structure PNPase from Streptomyces antibioticus was recently determined by x-ray crystallography. The enzyme is arranged in a homotrimeric multidomain complex with a central channel that could accommodate a single-stranded RNA chain. It was also observed that each PNPase subunit is composed of a duplicated structural core. In addition, the tungstate derivative structure reveals the PNPse active site in the second of these core domains (42, 43). Analysis of the biochemical properties of each domain of spinach chloroplast PNPase revealed unique features that may be related to the general function of RNA degradation (37). A distinctive feature of the S. antibioticus PNPase is that it can carry out pyrophosphate transfer from ATP to the 3'-OH of GTP to produce guanosine 3'-diphosphate 5'-triphosphate. Thus, S. antibioticus PNPase appears to be a bifunctional enzyme that possesses not only PNPase activity but also a guanosine pentaphosphate synthase activity that is not found for the E. coli PNPase (50, 51).

The decay mechanism of mRNA in E. coli cells has been extensively studied in many laboratories and involves a series of endo- and exolytic events. Several parallel pathways appear to exist that can partially substitute for each other. It was suggested that the decay of many mRNA species is initiated by the endonuclease, RNase E. The mRNA fragments resulting from RNase E cleavage are believed to be further degraded endonucleolytically and then cleaved by the 3' to 5' exonucleases, PNPase and RNase II (reviewed in Ref. 52). In E. coli cells, PNPase is mostly present in the cytoplasm (15), and about 10-20% of the enzyme population is associated with other proteins in a high molecular weight complex called the "RNA degradosome" (reviewed in Ref. 53). The major protein components identified in the complex include: PNPase, the DEAD-box RhlB RNA helicase, and the glycolytic enzyme enolase assembled on the C-terminal region of the endoribonuclease, RNase E. It was suggested that the components of the degradosome along with poly(A) polymerase may cooperate in the processing of mRNA (for recent references see Refs. 54 and 55). However, questions remain about whether the degradosome assembly is essential for bacterial mRNA decay in vivo (52, 56). It was also reported that chloroplast PNPase is not associated with other proteins to form a degradosome-like complex and appears to be a homomultimer complex of 600 kDa (36). Recent studies have indicated that polyadenylation is required for initiating exonucleolytic RNA degradation. It was shown that PNPase may function as an alternative poly(A) polymerase in E. coli cells where under appropriate conditions it can either degrade RNA or synthesize poly(A) tails, also incorporating C and U residues at low frequency in wild-type cells (57).

In 1968, I proposed to the management of the Weizmann Institute that they should initiate research in neurobiology and open a department devoted to this field. My interest in neuroscience developed slowly coinciding with the successful advances made in molecular biology and genetics. It became obvious that the time was ripe to apply similar interdisciplinary approaches to neurobiology. In October 1969, the NIH invited me to be their first Fogarty Scholar in Residence. This was an opportunity to learn more about neurobiology. I chose to join Marshall Nirenberg, Chief of the Laboratory of Biochemical Genetics at NHLBI, as his interests had also shifted from molecular biology to neurobiology. Together with Marshall, we organized a Fogarty Conference on Neuronal Plasticity. About a dozen leading molecular biologists participated in the lively discussions. I learned a great deal during my stay in Marshall's laboratory, and out of that I established, upon my return to Israel, the Department of Neurobiology at the Weizmann Institute and served as its Chairman until 1988. Marshall very generously allowed us to use in Rehovot the cloned cell lines he had isolated from a spontaneous mouse neuroblastoma tumor, C-1300 (58). Already during my stay in his laboratory, I devised methods for separating neurites from cell bodies that we later used in collaboration with Mary Catherine Glick (Children's Hospital of Philadelphia) to investigate their glycoprotein composition (59). At the start, I asked whether it would be possible to induce these cells to differentiate. Together with Yosef Kimhi, Clive Palfrey, and Ilan Spector it was found that dimethyl sulfoxide (Me2SO) is a potent inducer of differentiation of several of the mouse cell lines. Our results also suggested that the development of the excitable membrane could have taken place independently of the induction of neurospecific enzyme and did not require a sustained elevation of cyclic AMP levels (60). As with mouse neuroblastoma, we have found that several human cell lines could be induced to differentiate with different external biological response modifiers. In collaboration with Paul Marks (then at Columbia University) and colleagues, we demonstrated that hexamethylene bisacetamide (HMBA) is effective in inducing differentiation of mouse and human neuroblastoma cells at concentrations 50-fold lower than that of Me2SO, an observation that has clinical implications. The ability of some human cell lines to differentiate in vitro provides a model system to study the molecular events regulating cell growth and maturation of these neural crest derivatives and may provide insight into the mechanisms contributing to their tumorigenic conversion. Thus, with Mary Catherine Glick, we detected a 200-kDa glycoprotein that was associated with the surface membranes of mouse and human neuroblastoma cells displaying active Na+ channels (59). In other studies, Mona Soreq, Aliza Zutra, and Ruth Miskin observed significant levels of intracellular and secreted tissue-type plasminogen activator (tPA) activities in cultured mouse neuroblastoma cells, and upon differentiation with dibutyryl cyclic AMP the level of tPA increased about 20-fold (61). The high level of secreted tPA in neuroblastoma cells may perhaps be linked to recent results suggesting the involvement of plasmin in the processing of human amyloid precursor protein (reviewed in Ref. 62).

Because of the universal occurrence and importance of microtubules for neurite outgrowth, synapse formation, and axoplasmic transport, we decided to investigate the in vitro synthesis of tubulin, the major structural subunit protein of microtubules. The regulation of tubulin synthesis at both the transcriptional and translational levels was followed in these studies by Henri Schmitt (a visiting scientist from the Free University of Brussels) and Illana Gozes (then a graduate student and currently a Professor and Head of the Department of Clinical Biochemistry at Tel Aviv University). Subsequent studies by Illana led to the exciting discovery that brain {alpha}-tubulin and {beta}-tubulin display extensive microheterogeneity that was developmentally determined, increasing in the mature brain (63). In subsequent studies Irith Ginzburg (now a Professor at the Department of Neurobiology of the Weizmann Institute) and her colleagues isolated several cDNA clones bearing sequences coding for rat brain tubulin and actin (64). We used the tubulin and actin cDNA clones to study the control of mRNA expression in a number of cell systems. Irith also was a pioneer in the use of tubulin antisense oligodeoxyribonucleotides to prevent neurite extension in nerve growth factor-induced PC12 cells. The results showed that at least two tubulin isoforms are involved in neurite outgrowth (65, 66). In further studies we focused our efforts on the expression of microtubule-associated proteins (MAPs). Thus, I developed a specific binding assay that monitors the interaction of MAPs with tubulin cleavage peptides fixed to nitrocellulose membranes. To identify the tubulin-binding domains for MAPs we examined, in collaboration with Professor Herwig Ponstingl (from the German Cancer Research Center, Heidelberg) the binding of labeled rat brain MAP2 and tau factors to 60 cleavage peptides derived from pig {alpha}- and {beta}-tubulin. Both MAP2 and tau factors were found to specifically interact with peptides derived from the C-terminal region of {beta}-tubulin. The binding studies suggested that amino acid residues 434-440 of {beta}-tubulin are essential for the interaction of MAP2 and tau factors (67). It is interesting to note that the main amino acid sequence divergence of the various isotubulins and most of the post-translational modifications take place within their C-terminal regions. This led to the hypothesis that the variability in the C-terminal region will determine the strength and specificity of interaction of isotubulins with the various MAPs, thus generating functionally different microtubules. About that time, studies by Joachim Kirsch (a visiting scientist and currently Professor at the University of Heidelberg) revealed that neurite outgrowth is accompanied by reorganization of the microtubular cytoskeleton. Thus, upon neurite extension in neuroblastoma cells, microtubule bundles are formed in which MAP2 is found in the proximal part and in branching points. By contrast, MAP1B is distributed along the entire length of the neurite. The differences in spatial distribution between MAP1B and MAP2 along the neurites illustrate the heterogeneity in microtubule composition in various parts of the cell process and may suggest a different function for MAP1B (68).

We have also shown that differentiation of cultured human neuroblastoma cells requires priming with an inducer and subsequent interaction with components of the extracellular matrix (in particular, adhesion to laminin). The major binding site in laminin (mediating cell attachment) was then identified as containing the YIGSR peptide sequence. Affinity chromatography revealed one major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa laminin-binding protein (LBP) appeared to be down-regulated upon differentiation of human neuroblastoma cells (69). Cross-linking experiments indicated that in neuroblastoma cells the 67-kDa LBP binds to the YIGSR sequence in a cooperative manner through an association with another protein. About that time, Joachim Kirsch identified a new 250-kDa MAP2 isoform (designated MAP2d) in cultured human neuroblastoma cells. MAP2d was also observed in fetal human brain but was not found in adult human cerebellum (68). Analysis of human nerve cell-derived tumors showed that MAP2d is present in specimens from solid tumors diagnosed as neuroblastoma but hardly in ganglioneuroma tumors. In contrast both neuroblastoma and ganglioneuroma tumors contain significant levels of MAP1B. These studies indicated that MAP2d is associated with human embryonic nerve cells, as well as immature undifferentiated neuroblastoma tumors, and together with the 67-kDa LBP may lead to new prognostic tools for neuroblastoma tumors.

Other studies by Joachim Kirsch in collaboration with Professor Heinrich Betz and his colleagues at the Max-Planck-Institute for Brain Research, Frankfurt, have demonstrated that a 93-kDa glycine receptor-associated protein binds to tubulin. This 93-kDa protein (termed Gephyrin) appears to anchor the glycine receptor at post-synaptic sites via binding to subsynaptic tubulin and thus serves an important role in the topological organization of the post-synaptic membrane (70).

Address correspondence to: uri.littauer{at}weizmann.ac.il.



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N. Awano, M. Inouye, and S. Phadtare
RNase Activity of Polynucleotide Phosphorylase Is Critical at Low Temperature in Escherichia coli and Is Complemented by RNase II
J. Bacteriol., September 1, 2008; 190(17): 5924 - 5933.
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